U.S. patent number 6,696,238 [Application Number 09/917,340] was granted by the patent office on 2004-02-24 for transplant media.
Invention is credited to Jonathan F. McAnulty, Christopher J. Murphy, Ted W. Reid.
United States Patent |
6,696,238 |
Murphy , et al. |
February 24, 2004 |
Transplant media
Abstract
The present invention relates to media containing purified
antimicrobial polypeptides, such as defensins, and/or cell surface
receptor binding proteins. The media may also contain buffers,
macromolecular oncotic agents, energy sources, impermeant anions,
ATP substrates. The media find use for the storage and preservation
of internal organs prior to transplant.
Inventors: |
Murphy; Christopher J.
(Madison, WI), McAnulty; Jonathan F. (Oregon, WI), Reid;
Ted W. (Lubbock, TX) |
Family
ID: |
27396971 |
Appl.
No.: |
09/917,340 |
Filed: |
July 27, 2001 |
Current U.S.
Class: |
435/1.1; 435/1.2;
435/1.3 |
Current CPC
Class: |
A01N
1/0215 (20130101); A01N 1/0221 (20130101); A01N
1/021 (20130101) |
Current International
Class: |
A01N
1/02 (20060101); A01N 001/00 () |
Field of
Search: |
;435/1.1,1.2,1.3,2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
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Reid et al., IOVS 39:S78 (1998). .
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Segal et al., J. Infect. Diseases151:890-894 [1985]. .
Ganz et al., J. Clin. Invest. 76:1427-1435 [1985]. .
Wilde et al., J. Biol. Chem. 264:11200-11203 [1989]. .
Eisenhauer et al., Infection and Immunity 57:2021-2027 [1989].
.
Selsted et al., Infect. Immun. 55:2281-2286 [1987]. .
Merrifield et al., Ciba Found Symp. 186:5-26 (1994). .
Wade et al., Proc. Natl. Acad. Sci. USA 87(12):4761-5 (1990). .
Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156. .
Beaucage et al., Tetra Lett. 22:1859-1862 [1981]. .
Rein et al., Computer-Assisted Modeling of Receptor-Ligand
Interactions, Alan Liss, N.Y., [1989]. .
Reid et al., J. of Cellular Biochemistry 52:476-485
(1993)..
|
Primary Examiner: Saucier; Sandra E.
Attorney, Agent or Firm: Medlen & Carroll, LLP
Parent Case Text
This application claims priority to U.S. provisional application
No. 60/221,632, filed Jul. 28, 2000, No. 60/249,602, filed Nov. 17,
2000, and No. 60/290,932, filed May 15, 2001.
Claims
What is claimed is:
1. A composition comprising a purified antimicrobial polypeptide,
cell surface receptor binding compound and hydroxyethyl starch.
2. The composition of claim 1, wherein said purified antimicrobial
polypeptide and said hydroxyethyl starch are in solution.
3. The composition of claim 2, wherein said hydroxyethyl starch is
present in a concentration of about 1 to 200 g/l.
4. The composition of claim 1, wherein said purified antimicrobial
polypeptide is a purified defensin.
5. The composition of claim 4, wherein said purified defensin is
present in a concentration of about 0.01 to 1000 mg/l.
6. The composition of claim 4, wherein said purified defensin is
present in a concentration of about 0.1 to 5 mg/l.
7. The composition of claim 4, wherein said purified defensin is
present in a concentration of about 0.01 to 1000 mg/l and said
hydroxyethyl starch is present in a concentration of about 1 to 200
g/l.
8. The composition of claim 4, wherein said defensin is encoded by
SEQ ID NO:37.
9. The composition of claim 1, wherein said cell surface receptor
binding compound is selected from the group consisting of IGF-1,
EGF, NGF, and substance P and combinations thereof.
10. A composition comprising a purified defensin and an impermeant
anion selected from the group consisting of lactobionate and
gluconate.
11. The composition of claim 10, wherein said antimicrobial
polypeptide and said impermeant ion are in solution.
12. The composition of claim 10, wherein said purified defensin is
present in a concentration of about 0.01 to 1000 mg/l.
13. The composition of claim 10, wherein said impermeant ion is
lactobionic acid, and wherein said lactobionate is present in a
concentration of about 1 to 500 mM.
14. The composition of claim 10, wherein said impermeant anion is
gluconate, and wherein said gluconate is present in a concentration
of about 1 to 500 mM.
15. The composition of claim 10, wherein said defensin is encoded
by SEQ ID NO:37.
16. The composition of claim 10, further comprising a cell surface
receptor binding compound.
17. The composition of claim 16, wherein said cell surface receptor
binding compound is selected from the group consisting of IGF-1,
EGF, NGF, and substance P and combinations thereof.
Description
FIELD OF THE INVENTION
The present invention relates to media comprising purified
antimicrobial peptides, pore forming agents, and/or cell surface
receptor binding compounds and their use for the storage and
preservation of organs prior to transplant.
BACKGROUND OF THE INVENTION
A wide variety of organs, including kidneys, lungs, livers, hearts,
pancreases, and small intestines are routinely and successfully
transplanted. These organs are obtained either from living donors
or from cadaveric sources.
In 1998, a total of 12,166 kidney transplants were performed in the
United States by programs tracked by the UNOS Transplant Patient
DataSource. A total of 45,189 people were on the waiting lists for
kidneys as of Sep. 30, 1999. Over 20,000 kidneys were transplanted
between Jul. 1, 1995 and Jun. 30, 1997. The graft survival rate for
these 2(transplanted kidneys was 93.4% after three months.
The ability to store organs for two or three days prior to
transplantation allows sufficient time for histo-compatibility
testing of donor and recipient, transport of the organ between
transplant centers, preoperative preparation of the recipient,
preliminary donor culture testing, and vascular repair of the organ
if needed. The efficacy of organ transplantation depends in part on
how well the organ is preserved prior to transplantation. Two
methods are used to preserve organs prior to transplant:
hypothermic storage and continuous pulsatile perfusion. Hypothermic
storage by simple cold storage methods involves removal of an organ
from a donor followed by rapid cooling. Cooling is achieved by a
combination of external cooling and a short period of perfusion
with a chilled medium to reduce the core temperature of the organ
as quickly as possible. The organs are then immersed in a flush-out
medium at from 0.degree. C. to 4.degree. C. Continuous pulsatile
perfusion involves the continuous infusion of organs with a
preservation solution designed to prevent low temperature
injury.
A number of media have been developed for infusing and preserving
organs prior to transplantation. Examples of such media include
VIASPAN (also known as University of Wisconsin solution; Barr
Laboratories, Pomona, N.Y.), University of Wisconsin Machine
Perfusion Solution, Hypertonic Citrate Solution, HTK Solution, HTK
Solution of Bretschneider, Phosphate Buffered Sucrose, EuroCollins
Solution, and Collins C2 Solution. However, none of these media are
able to extend the preservation of organs past about 72 hours using
cold storage methods. Additional preservation time would be useful
for tests and for transportation of the organs. Furthermore, media
that increase preservation time also can be expected to provide
healthier organs for transplants performed within 72 hours.
Accordingly, what is needed in the art are improved media for
preserving and storing organs prior to transplant. Such media
should be able to extend the preservation period past 72 hours and
provide organs with increased functionality upon transplant.
SUMMARY OF THE INVENTION
The present invention relates to media comprising antimicrobial
polypeptides or pore forming agents and/or cell surface receptor
binding compounds and their use for the storage and preservation of
organs prior to transplant.
The present invention is not limited to any particular media or
formulation. Indeed, a variety of medias and formulations are
contemplated. In some embodiments, the present invention provides
compositions comprising a purified antimicrobial polypeptide and
hydroxyethyl starch. The present invention is not limited to any
particular antimicrobial peptide. Indeed a variety of antimicrobial
peptides are contemplated, including, but not limited to, those
identified by SEQ ID NOs:1-96. In some preferred embodiments, the
antimicrobial peptide is a defensin. The present invention is not
limited to any particular defensin. Indeed, the use of a variety of
defensins is contemplated, including, but not limited to those
identified by SEQ ID NOs:37-96. In particularly preferred
embodiments, the antimicrobial peptide is bovine dodecapeptide or
BNP-1 (SEQ ID NO: 37). In some preferred embodiments, the
antimicrobial polypeptide or defensin comprises D-amino acids. In
some embodiments, the antimicrobial peptide and hydroxyethyl starch
are in solution. The media of the present invention are not limited
to any particular concentration of antimicrobial peptide. Indeed, a
range of concentrations are contemplated (e.g., from about 0.01 to
1000 mg/l and preferably from about 0.1 to 5 mg/1). The present
invention is not limited to any particular concentration of
hydroxyethyl starch. Indeed, a range of concentrations are
contemplated (e.g., from about 1 to 200 g/l). In some embodiments,
the media further comprises a cell surface receptor binding
compound. The present invention is not limited to any particular
cell surface receptor binding compound. Indeed, a variety of cell
surface receptor binding compounds are contemplated, including, but
not limited to IGF-1, EGF, NGF, and substance P.
In other embodiments, the present invention provides compositions
comprising an antimicrobial polypeptide and an impermeant anion
selected from the group consisting of lactobionic acid and
gluconate. In some preferred embodiments, the antimicrobial
polypeptide and the impermeant ion are in solution. The present
invention is not limited to any particular antimicrobial peptide.
Indeed a variety of antimicrobial peptides are contemplated,
including, but not limited to, those identified by SEQ ID NOs:1-96.
In some preferred embodiments, the antimicrobial peptide is a
defensin. The present invention is not limited to any particular
defensin. Indeed, the use of a variety of defensins is
contemplated, including, but not limited to those identified by SEQ
ID NOs:37-96. In some preferred embodiments, the antimicrobial
polypeptide or defensin comprises D-amino acids. In particularly
preferred embodiments, the antimicrobial peptide is bovine
dodecapeptide or BNP-1 (SEQ ID NO: 37). The media of the present
invention are not limited to any particular concentration of
antimicrobial peptide. Indeed, a range of concentrations are
contemplated (e.g., from about 0.01 to 1000 mg/l and preferably
from about 0.1 to 5 mg/l). The media of the present invention are
not limited to any particular concentration of impermeant ion.
Indeed, a range of concentrations are contemplated (e.g., from
about 1 to 500 mM). In some embodiments, the media further
comprises a cell surface receptor binding compound. The present
invention is not limited to any particular cell surface receptor
binding compound. Indeed, a variety of cell surface receptor
binding compounds are contemplated, including, but not limited to
IGF-1, EGF, NGF, and substance P. In some preferred embodiments,
the media does not require the use of hydroxyethyl starch.
In other embodiments, the present invention provides compositions
comprising an antimicrobial polypeptide and glutathione. In some
preferred embodiments, the antimicrobial polypeptide and the
impermeant ion are in solution. The present invention is not
limited to any particular antimicrobial peptide. Indeed a variety
of antimicrobial peptides are contemplated, including, but not
limited to, those identified by SEQ ID NOs:1-96. In some preferred
embodiments, the antimicrobial peptide is a defensin. The present
invention is not limited to any particular defensin. Indeed, the
use of a variety of defensins is contemplated, including, but not
limited to those identified by SEQ ID NOs:37-96. In some preferred
embodiments, the antimicrobial polypeptide or defensin comprises
D-amino acids. In particularly preferred embodiments, the
antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO:
37). The media of the present invention are not limited to any
particular concentration of antimicrobial peptide. Indeed, a range
of concentrations are contemplated (e.g., from about 0.01 to 1000
mg/l and preferably from about 0.1 to 5 mg/l). The media of the
present invention are not limited to any particular concentration
of glutathione. Indeed, a range of concentrations are contemplated
(e.g., from about 0.1 to 100 mM). In some embodiments, the media
further comprises a cell surface receptor binding compound. The
present invention is not limited to any particular cell surface
receptor binding compound. Indeed, a variety of cell surface
receptor binding compounds are contemplated, including, but not
limited to IGF-1, EGF, NGF, and substance P. In some preferred
embodiments, the media does not require the use of hydroxyethyl
starch.
In further embodiments, the present invention provides compositions
comprising a purified antimicrobial polypeptide and an ex vivo
internal organ. The present invention is not limited to any
particular internal organ. Indeed, a variety of internal organs are
contemplated, including, but not limited to kidneys, hearts, lungs,
small intestines, large intestines, livers, and pancreases. The
present invention is not limited to organs from any particular
species of animal. Indeed, use of organs from a variety of animals
is contemplated, including organs from humans, pigs, and dogs. The
present invention is not limited to any particular antimicrobial
peptide. Indeed a variety of antimicrobial peptides are
contemplated, including, but not limited to, those identified by
SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial
peptide is a defensin. The present invention is not limited to any
particular defensin. Indeed, the use of a variety of defensins is
contemplated, including, but not limited to those identified by SEQ
ID NOs:37-96. In particularly preferred embodiments, the
antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO:
37). In some preferred embodiments, the antimicrobial polypeptide
or defensin comprises D-amino acids. The media of the present
invention are not limited to any particular concentration of
antimicrobial peptide. Indeed, a range of concentrations are
contemplated (e.g., from about 0.01 to 1000 mg/l and preferably
from about 0.1 to 5 mg/l). In some embodiments, the compositions
further comprise a macromolecular oncotic agent. The present
invention is not limited to any particular macromolecular oncotic
agent. Indeed, a variety of macromolecular oncotic agents are
contemplated, including, but not limited to hydroxyethyl starch,
dextran, and glucose. In other embodiments, the composition further
comprises an impermeant anion. The present invention is not limited
to any particular impermeant anion. Indeed, a variety of impermeant
anions are contemplated, including, but not limited to, gluconate
and lactobionic acid. In still further embodiments, the
compositions comprise glutathione. In some embodiments, the
compositions further comprise a cell surface receptor binding
compound. The present invention is not limited to any particular
cell surface receptor binding compound. Indeed, a variety of cell
surface receptor binding compounds are contemplated, including, but
not limited to IGF-1, EGF, NGF, and substance P. In some preferred
embodiments, the media does not require the use of hydroxyethyl
starch.
In still other embodiments, the present invention provides methods
comprising a) providing cellular material and a solution comprising
a purified antimicrobial polypeptide and b) storing the cellular
material in said solution comprising a purified antimicrobial
peptide. The present invention is not limited to the storage of any
particular cellular material. Indeed, a variety of cellular
materials are contemplated, including but not limited to internal
organs, skin, and gametes. In some preferred embodiments, the
cellular material is an internal organ. The present invention is
not limited to any particular internal organ. Indeed, a variety of
internal organs are contemplated, including, but not limited to
kidneys, hearts, lungs, small intestines, large intestines, livers,
and pancreases. The present invention is not limited to organs from
any particular species of animal. Indeed, use of organs from a
variety of animals is contemplated, including organs from humans,
pigs, and dogs. In some embodiments, the internal organ is infused
with the solution. The present invention is not limited to any
particular antimicrobial peptide. Indeed a variety of antimicrobial
peptides are contemplated, including, but not limited to, those
identified by SEQ ID NOs:1-96. In some preferred embodiments, the
antimicrobial peptide is a defensin. The present invention is not
limited to any particular defensin. Indeed, the use of a variety of
defensins is contemplated, including, but not limited to those
identified by SEQ ID NOs:37-96. In particularly preferred
embodiments, the antimicrobial peptide is bovine dodecapeptide or
BNP-1 (SEQ ID NO: 37). In some preferred embodiments, the
antimicrobial polypeptide or defensin comprises D-amino acids. The
media of the present invention are not limited to any particular
concentration of antimicrobial peptide. Indeed, a range of
concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l
and preferably from about 0.1 to 5 mg/l). In some embodiments, the
compositions further comprise a macromolecular oncotic agent. The
present invention is not limited to any particular macromolecular
oncotic agent. Indeed, a variety of macromolecular oncotic agents
are contemplated, including, but not limited to hydroxyethyl
starch, dextran, and glucose. In other embodiments, the composition
further comprises an impermeant anion. The present invention is not
limited to any particular impermeant anion. Indeed, a variety of
impermeant anions are contemplated, including, but not limited to,
gluconate and lactobionic acid. In still further embodiments, the
compositions comprise glutathione. In some embodiments, the
compositions further comprise a cell surface receptor binding
compound. The present invention is not limited to any particular
cell surface receptor binding compound. Indeed, a variety of cell
surface receptor binding compounds are contemplated, including, but
not limited to IGF-1, EGF, NGF, and substance P. In some preferred
embodiments, the media does not require the use of hydroxyethyl
starch.
In still further embodiments, the present invention provides
compositions comprising a cell surface receptor binding compound
and hydroxyethyl starch. The present invention is not limited to
any particular cell surface receptor binding compound. Indeed, a
variety of cell surface receptor binding compounds are
contemplated, including, but not limited to IGF-1,EGF, NGF, and
substance P.
In other embodiments, the present invention provides compositions
comprising a cell surface receptor binding compound and an internal
organ. In some embodiments, the compositions further comprise a
macromolecular oncotic agent. The present invention is not limited
to any particular macromolecular oncotic agent. Indeed, a variety
of macromolecular oncotic agents are contemplated, including, but
not limited to hydroxyethyl starch, dextran, and glucose. In other
embodiments, the composition further comprises an impermeant anion.
The present invention is not limited to any particular impermeant
anion. Indeed, a variety of impermeant anions are contemplated,
including, but not limited to, gluconate and lactobionic acid. In
still further embodiments, the compositions comprise glutathione.
In some preferred embodiments, the media does not require the use
of hydroxyethyl starch.
In some embodiments, the present invention provides compositions
comprising trehalose and hydroxyethyl starch. In some preferred
embodiments, the trehalose and hydroxyethyl starch are in solution.
The present invention is not limited to any particular
concentration of trehalose. Indeed, a range of concentrations are
contemplated (e.g., from about 1 mM to 30 mM). In some embodiments,
the compositions further comprise an antimicrobial peptide and/or
cell surface receptor binding compound. In some embodiments, the
compositions further comprise a cell surface receptor binding
compound. The present invention is not limited to any particular
cell surface receptor binding compound. Indeed, a variety of cell
surface receptor binding compounds are contemplated, including, but
not limited to IGF-1, EGF, NGF, and substance P. The present
invention is not limited to any particular antimicrobial peptide.
Indeed a variety of antimicrobial peptides are contemplated,
including, but not limited to, those identified by SEQ ID NOs:1-96.
In some preferred embodiments, the antimicrobial peptide is a
defensin. The present invention is not limited to any particular
defensin. Indeed, the use of a variety of defensins is
contemplated, including, but not limited to those identified by SEQ
ID NOs:37-96. In particularly preferred embodiments, the
antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO:
37). The media of the present invention are not limited to any
particular concentration of antimicrobial peptide. Indeed, a range
of concentrations are contemplated (e.g., from about 0.01 to 1000
mg/l and preferably from about 0.1 to 5 mg/l). In some embodiments,
the compositions further comprise a macromolecular oncotic agent.
The present invention is not limited to any particular
macromolecular oncotic agent. Indeed, a variety of macromolecular
oncotic agents are contemplated, including, but not limited to
hydroxyethyl starch, dextran, and glucose. In other embodiments,
the composition further comprises an impermeant anion. The present
invention is not limited to any particular impermeant anion.
Indeed, a variety of impermeant anions are contemplated, including,
but not limited to, gluconate and lactobionic acid. In still
further embodiments, the compositions comprise glutathione.
In other embodiments, the present invention provides a kit
comprising a vessel containing a solution comprising a compound
selected from the group consisting of lactobionate and hydroxyethyl
starch; and a vessel containing an antimicrobial polypeptide. In
some embodiments, the antimicrobial polypeptide is BNP-1. In other
embodiments, the vessel containing an antimicrobial polypeptide
further comprises a cell surface receptor binding compound. In
further embodiments, the cell surface receptor binding compound is
selected from the group consisting of IGF-1, EGF, NGF, and
substance P. In some embodiments, the kit further comprises
instructions for combining said solution and the antimicrobial
polypeptide.
In still further embodiments, the present invention provides
processes for producing a storage solution comprising providing a
solution comprising a compound selected from the group consisting
of hydroxyethyl starch and lactobionate and a purified
antimicrobial polypeptide; and combining said solution with the
purified antimicrobial polypeptide. In some embodiments, the method
further comprising the steps of providing at least one cell surface
receptor binding compound and combining the at least one cell
surface receptor binding compound with the solution and the
antimicrobial polypeptide.
In some preferred embodiments, the present invention provides a
composition comprising hydroxyethyl starch or lactobionate and an
antimicrobial polypeptide for use as an organ storage or perfusion
solution. In some embodiments, the composition further comprising a
cell surface receptor binding compound. In other preferred
embodiments, the present invention provides a composition
comprising a purified antimicrobial polypeptide (e.g., BNP-1) and
at least one purified cell surface receptor binding compound (e.g.,
IGF-1, EGF, NGF, and substance P), for use as a supplement for
organ storage solutions.
In some embodiments, the media described herein further comprise a
microtubule stabilizing agent selected from the group consisting of
taxol, discodermolide, epothilone A and B, vinblastine, and
vinchristine.
In still further embodiments, the present invention provides
methods and compositions for stabilizing microtubules in cells,
tissues, or organs, either in vitro, in vivo, or ex vivo. In
preferred embodiments, the compositions comprise a defensin (e.g.,
BNP-1). In other preferred embodiments, the compositions comprise a
cell surface receptor binding compound, impermeant anion, energy
source, or macromolecular oncotic agent as described in more detail
above. In other particularly preferred embodiments, the present
invention provides a composition comprising a defensin (e.g.,
BNP-1) for use in stabilizing microtubules and/or actin filaments.
In still other embodiments, the present invention provides methods
and processes comprising providing a cell, tissue or organ, and a
composition comprising a purified defensin, and treating the cell,
tissue, or organ under conditions such that the cytoskeleton of the
cell tissue, or organ is stabilized. In particularly preferred
embodiments, microtubules and and/or actin filaments are
stabilized. In still other particularly preferred embodiments, the
defensin id BNP-1 (SEQ ID NO: 37).
In still further embodiments, the present invention provides a
composition substantially as described in any of the examples
herein.
DESCRIPTION OF THE FIGURES
FIG. 1 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for 3 days in UW
solution alone (solid line) or in UW solution supplemented with
BNP-1 (dashed line).
FIG. 2 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for four days in UW
solution alone (solid circles), in UW solution supplemented with
BNP-1 (solid squares), or in UW solution supplemented with BNP-1
and growth factors (x's).
FIG. 3 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for four days in UW
solution alone (solid triangles) or six days in UW solution
supplemented with trophic factors (unfilled triangles).
FIG. 4 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for three days in UW
solution alone (solid tangles) or six days in UW solution
supplemented with trophic factors (squares).
FIG. 5 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for three days in UW
solution alone (squares) or five days in UW solution supplemented
with trophic factors (circles).
FIG. 6 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for three days in UW
solution alone (squares) or four days in UW solution supplemented
with trophic factors (diamonds).
FIG. 7 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for four days in UW
solution alone (solid triangles) or four days in UW solution
supplemented with trophic factors (diamonds).
FIG. 8 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for five days in UW
solution with trophic factors and with starch (circles) or five
days in UW solution supplemented with trophic factors and without
starch (squares).
FIG. 9 is a graph showing serum creatinine levels (Y-axis) over
time (X-axis) in dogs receiving kidneys stored for three days in UW
solution supplemented with BNP-1 (L-form isomer)(circles) or three
days in UW solution supplemented with BNP-1 (D-form isomer)
(squares).
DEFINITIONS
To facilitate understanding of the invention, a number of terms are
defined below.
As used herein, the term "antimicrobial polypeptide" refers to
polypeptides that inhibit the growth of microbes (e.g., bacteria).
Examples of antimicrobial polypeptides include, but are not limited
to, the polypeptides described in Table 1 below (e.g., defensins).
Antimicrobial polypeptides include peptides synthesized from both
L-amino and D-amino acids.
As used herein, the term "pore forming agent" refers to any agent
(e.g., peptide or other organic compound) that forms pores in a
biological membrane. When the pore forming agent is a peptide, the
peptide can be synthesized from both L-amino and D-amino acids.
As used herein, the term "cell surface receptor binding compound"
refers to any compound that directly or indirectly (e.g., binding
through an intermediate agent) binds to a cell surface receptor
(e.g., an agonist). Cell surface receptor binding compounds can be
proteins (e.g., IGF-1 [insulin-like growth factor 1], IGF-2
[insulin-like growth factor 2], NGF-.beta. [nerve growth
factor-.beta.], EGF [epidermal growth factor], CSGF
[colony-stimulating growth factor], FGF [fibroblast growth factor],
PDGF [platelet-derived growth factor], VEGF [vascular endothelial
growth factor], TGF-.beta. [transforming growth factor .beta.], and
bone morphogenetic proteins), either purified from natural sources
or genetically engineered, as well as fragments, mimetics,
derivatives or modifications thereof, and other organic compounds
that bind to cell surface receptors (e.g., prostaglandins). Further
examples of cell surface receptor binding compounds are provided in
U.S. Pat. Nos. 5,183,805; 5,218,093; 5,130,298; 5,639,664;
5,457,034; 5,210,185; 5,470828; 5,650,496; 5,998,376; and
5,410,019; all of which are incorporated herein by reference.
As used herein, the term "cellular material" refers to any material
or composition comprising cells (e.g., cultured cells, gametes
(i.e., sperm and eggs), embryos, tissues, organs, and
organisms).
As used herein, the term "internal organ" refers to an organ
located in the interior of the body (e.g., in the thoracic or
abdominal cavity). Examples of internal organs include, but are not
limited to kidneys, hearts, lungs, small intestines, large
intestines, livers, and pancreases. Internal organs can be provided
from a human donor (either cadaveric or living) or be from an
animal (e.g., for xenotransplants or transplant studies in an
animal model such as dogs).
As used herein, the term "delayed graft function" refers to the
delay in the return to normal serum creatinine following kidney
transplant.
As used herein, the term "impermeant anion" refers to compounds
that counteract swelling in organs that have been exposed to
hypothermic temperatures. Examples of impermeant anions include,
but are not limited to, gluconate and lactobionic acid.
As used herein, the term "macromolecular oncotic agent" refers to
compounds used to maintain oncotic pressure equivalent to that of
blood plasma. Examples of macromolecular oncotic agents include,
but are not limited to, hydroxyethyl starch, dextran, trehalose,
raffinose, mannitol, sucrose and glucose.
The term "recombinant protein" or "recombinant polypeptide" as used
herein refers to a protein molecule expressed from a recombinant
DNA molecule. In contrast, the term "native protein" or "native
polypeptide" is used herein to indicate a protein isolated from a
naturally occurring (i.e., a nonrecombinant) source. Molecular
biological techniques may be used to produce a recombinant form of
a protein or polypeptide with similar or identical properties as
compared to the native form of the protein.
Where "amino acid sequence" is recited herein to refer to an amino
acid sequence of a naturally occurring protein molecule, "amino
acid sequence" and like terms, such as "polypeptide" or "protein"
are not meant to limit the amino acid sequence to the complete,
native amino acid sequence associated with the recited protein
molecule.
As used herein in reference to an amino acid sequence or a protein,
the term "portion" (as in "a portion of an amino acid sequence")
refers to fragments of that protein. The fragments may range in
size from four amino acid residues to the entire amino acid
sequence minus one amino acid (e.g., 5, 6, 7, 8, . . . x-1).
As used herein, the term "variant," when used in reference to a
protein, refers to proteins encoded by partially homologous nucleic
acids so that the amino acid sequence of the proteins varies. As
used herein, the term "variant" encompasses proteins encoded by
homologous genes having both conservative and nonconservative amino
acid substitutions that do not result in a change in protein
function, as well as proteins encoded by homologous genes having
amino acid substitutions that cause decreased protein function or
increased protein function.
As used herein, the term "fusion protein" refers to a chimeric
protein containing the protein of interest (e.g., defensins and
fragments thereof) joined to a heterologous protein fragment (e.g.,
the fusion partner which consists of a non-defensin protein). The
fusion partner may enhance the solubility of a defensin as
expressed in a host cell, may provide an affinity tag to allow
purification of the recombinant fusion protein from the host cell
or culture supernatant, or both. If desired, the fusion protein may
be removed from the protein of interest (e.g., defensin or
fragments thereof) by a variety of enzymatic or chemical means know
to the art.
As used herein, the term "purified" refers to molecules, either
nucleic or amino acid sequences, that are removed from their
natural environment, isolated or separated. The percent of a
purified component is thereby increased in the sample. For example,
an "isolated defensin" is therefore a purified defensin.
"Substantially purified" molecules are at least 60% free,
preferably at least 75% free, and more preferably at least 90% free
from other components with which they are naturally associated.
The term "gene" as used herein, refers to a DNA sequence that
comprises control and coding sequences necessary for the production
of a polypeptide or protein precursor. The polypeptide can be
encoded by a full length coding sequence or by any portion of the
coding sequence, as long as the desired protein activity is
retained.
The term "homology" refers to a degree of complementarity. There
may be partial homology or complete homology (i.e., identity). A
partially complementary sequence is one that at least partially
inhibits a completely complementary sequence from hybridizing to a
target nucleic acid. This situation is referred to using the
functional term "substantially homologous." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous to a target under
conditions of low stringency. This is not to say that conditions of
low stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to
one another be a specific (i.e., selective) interaction. The
absence of non-specific binding may be tested by the use of a
second target which lacks even a partial degree of complementarity
(e.g., less than about 30% identity). In this case, in the absence
of non-specific binding, the probe will not hybridize to the second
non-complementary target.
When used in reference to a double-stranded nucleic acid sequence
such as a cDNA or a genomic clone, the term "substantially
homologous" refers to any probe which can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described herein.
As used herein, the term "hybridization" is used in reference to
the pairing of complementary nucleic acid strands. Hybridization
and the strength of hybridization (i.e., the strength of the
association between nucleic acid strands) is impacted by many
factors well known in the art including the degree of
complementarity between the nucleic acids, stringency of the
conditions involved affected by such conditions as the
concentration of salts, the T.sub.m (melting temperature) of the
formed hybrid, the presence of other components (e.g., the presence
or absence of polyethylene glycol), the molarity of the hybridizing
strands and the G:C content of the nucleic acid strands.
As used herein, the term "stringency" is used in reference to the
conditions of temperature, ionic strength, and the presence of
other compounds, under which nucleic acid hybridizations are
conducted. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences. Thus, conditions of
"medium" or "low" stringency are often required when it is desired
that nucleic acids which are not completely complementary to one
another be hybridized or annealed together. The art knows well that
numerous equivalent conditions can be employed to comprise medium
or low stringency conditions. The choice of hybridization
conditions is generally evident to one skilled in the art and is
usually guided by the purpose of the hybridization, the type of
hybridization (DNA-DNA or DNA-RNA), and the level of desired
relatedness between the sequences (e.g., Sambrook et al., 1989,
Nucleic Acid Hybridization, A Practical Approach, IRL Press,
Washington D.C., 1985, for a general discussion of the state of the
art).
The stability of nucleic acid duplexes is known to decrease with an
increased number of mismatched bases, and further to be decreased
to a greater or lesser degree depending on the relative positions
of mismatches in the hybrid duplexes. Thus, the stringency of
hybridization can be used to maximize or minimize stability of such
duplexes. Hybridization stringency can be altered by: adjusting the
temperature of hybridization; adjusting the percentage of helix
destabilizing agents, such as formamide, in the hybridization mix;
and adjusting the temperature and/or salt concentration of the wash
solutions. For filter hybridizations, the final stringency of
hybridizations often is determined by the salt concentration and/or
temperature used for the post-hybridization washes.
"High stringency conditions" when used in reference to nucleic acid
hybridization comprise conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of 5.times.
SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2 PO.sub.4.H.sub.2 O and 1.85
g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.
Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA
followed by washing in a solution comprising 0.1.times. SSPE, 1.0%
SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
"Medium stringency conditions" when used in reference to nucleic
acid hybridization comprise conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of 5.times.
SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2 PO.sub.4.H.sub.2 O and 1.85
g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.
Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA
followed by washing in a solution comprising 1.0.times. SSPE, 1.0%
SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
"Low stringency conditions" comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times. SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2 PO.sub.4.H.sub.2
O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times. Denhardt's reagent [50.times. Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)]
and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in
a solution comprising 5.times. SSPE, 0.1% SDS at 42.degree. C. when
a probe of about 500 nucleotides in length is employed.
As used herein, the term "T.sub.m " is used in reference to the
"melting temperature". The melting temperature is the temperature
at which 50% of a population of double-stranded nucleic acid
molecules becomes dissociated into single strands. The equation for
calculating the T.sub.m of nucleic acids is well-known in the art.
The T.sub.m of a hybrid nucleic acid is often estimated using a
formula adopted from hybridization assays in 1 M salt, and commonly
used for calculating T.sub.m for PCR primers: [(number of
A+T).times.2.degree. C.+(number of G+C).times.4.degree. C.]. (C. R.
Newton et al., PCR, 2nd Ed., Springer-Verlag (New York, 1997), p.
24). This formula was found to be inaccurate for primers longer
than 20 nucleotides. (Id.) Another simple estimate of the T.sub.m
value may be calculated by the equation: T.sub.m =81.5+0.41(% G+C),
when a nucleic acid is in aqueous solution at 1 M NaCl. (e.g.,
Anderson and Young, Quantitative Filter Hybridization, in Nucleic
Acid Hybridization (1985). Other more sophisticated computations
exist in the art which take structural as well as sequence
characteristics into account for the calculation of T.sub.m. A
calculated T.sub.m is merely an estimate; the optimum temperature
is commonly determined empirically.
As used herein, the term "vector" is used in reference to nucleic
acid molecules that transfer DNA segment(s) from one cell to
another and capable of replication in a cell. Vectors may include
plasmids, bacteriophages, viruses, cosmids, and the like.
The terms "recombinant vector" and "expression vector" as used
herein refer to DNA or RNA sequences containing a desired coding
sequence and appropriate DNA or RNA sequences necessary for the
expression of the operably linked coding sequence in a particular
host organism. Prokaryotic expression vectors include a promoter, a
ribosome binding site, an origin of replication for autonomous
replication in host cells and possibly other sequences, e.g., an
optional operator sequence. A promoter is defined as a DNA sequence
that directs RNA polymerase to bind to DNA and to initiate RNA
synthesis. Eukaryotic expression vectors include a promoter,
polyadenlyation signal and optionally an enhancer sequence.
As used herein the term "coding region" when used in reference to
structural gene refers to the nucleotide sequences which encode the
amino acids found in the nascent polypeptide as a result of
translation of a mRNA molecule. Typically, the coding region is
bounded on the 5' side by the nucleotide triplet "ATG" which
encodes the initiator methionine and on the 3' side by a stop codon
(e.g., TAA, TAG, TGA). In some cases the coding region is also
known to initiate by a nucleotide triplet "TTG".
The terms "buffer" or "buffering agents" refer to materials which
when added to a solution, cause the solution to resist changes in
pH.
The term "monovalent salt" refers to any salt in which the metal
(e.g., Na, K, or Li) has a net 1+ charge in solution (i.e., one
more proton than electron).
The term "divalent salt" refers to any salt in which a metal (e.g.,
Mg, Ca, or Sr) has a net 2+ charge in solution.
The term "solution" refers to an aqueous mixture.
The term "buffering solution" refers to a solution containing a
buffering reagent.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to media comprising antimicrobial
polypeptides and/or cell surface receptor binding compounds and
their use for the storage and preservation of organs prior to
transplant, and indeed, the preservation and storage of cellular
materials in general. The media provided herein are superior to
previously described media for organ preservation. Animals
receiving kidneys stored in the media of the present invention for
either three or four days had serum creatinine levels of less than
half of those observed in control animals receiving kidneys stored
in UW solution alone. Therefore, it is contemplated that the use of
the media of the present invention to preserve organs prior to
transplant results both in improved function of the organ after
transplant and an increase in the length of time for which the
organs can be stored (i.e., increased storage capability).
Lowered serum creatinine levels are indicative of healthier kidneys
and a more preferable prognosis for the transplant patient. It is
contemplated that transplant of healthier organs leads to a
decrease in chronic rejection. Chronic rejection is a host versus
graft rejection that occurs over a period of months to years, and
is characterized by arterial and arteriolar thickening, atrophy,
and fibrosis. Chronic rejection is the most common type of
rejection for most solid organ allografts. In fact, approximately
ten percent of kidney transplants fail each year due to chronic
rejection. A 1999 survey indicates that a majority of transplant
physicians and surgeons believe that chronic rejection is the area
of transplant medicine that needs the most improvement
(www.kidney.org/general/news/survey.cfm).
Additionally, use of the media of the present invention for cold
storage or machine perfusion is expected to greatly reduce costs
associated with delayed graft function in kidneys. Most kidney
transplant centers currently experience delayed graft function
rates of between 20% and 30%. When kidneys from non-beating heart
donors are utilized, the rate of delayed graft function increases
to approximately 75%-90%. Delayed graft function has been estimated
to add up to $20,000.00 to the cost of a kidney transplant due to
dialysis, complications, and longer hospital stay. Furthermore, the
incidence of delayed graft function is correlated with chronic
rejection (i.e., 53% of kidneys in patients that need dialysis
survive 5 years vs. 80% in optimal kidneys). The experimental data
provided below in the Examples demonstrates that use of the media
compositions of the present invention greatly reduces the time
required to return to normal serum creatinine levels and thus
reduces the incidence of delayed graft function.
Furthermore, it is expected that the media of the present invention
will also be useful for the storage and/or resuscitation of kidneys
from non-beating heart donors so that they can routinely be used
for transplant. As described above, the delayed graft function
rates associated with kidneys from non-beating heart donors exceeds
75%. The major source of delayed graft function of these kidneys is
believed to be warm ischemic injury. Most cold storage methods have
been completely unsuccessful in reducing preservation injury and
delayed graft function. As a result, kidneys from non-beating heart
donors that are subject to warm ischemic injury represent the
largest untapped source of donor kidneys. It is contemplated that
the use of the media of the present invention will facilitate
routine use of kidneys from non-beating heart donors, thus greatly
expanding the pool of kidneys available to recipients. In
particular, the use of the media of the present invention to store
kidneys from non-beating heart donors will result in a decrease in
the delayed graft function rates normally observed when those
kidneys are utilized for transplant.
Accordingly, improved compositions and methods for organ transplant
are described in detail below.
I. Transplant Media
The present invention contemplates the addition of antimicrobial
polypeptides (e.g., defensins) and/or cell surface receptor binding
compounds to media used for organ transplantation and other
procedures such as cardioplegia. In Section A, antimicrobial
peptides useful in the media of the present invention are
described. In Section B, cell surface receptor binding compounds
useful in the present invention are described. In Section C, other
components of organ transplantation media are described and
representative formulas for organ preservation media are
provided.
A. Antimicrobial Peptides
In some embodiments of the present invention, compositions for
preserving organs prior to transplantation are provided. In some
embodiments of the present invention, media for preserving organs
comprise one or more antimicrobial polypeptides (e.g.,
Antimicrobial Peptide Protocols, ed. W. M. Shafer, Humana Press,
Totowa, N.J. [1997]) or pore forming agents. In some embodiments,
the antimicrobial peptide or pore forming agent is a compound or
peptide selected from the following: magainin (e.g., magainin I,
magainin II, xenopsin, xenopsin precursor fragment, caerulein
precursor fragment), magainin I and II analogs (PGLa, magainin A,
magainin G, pexiganin, Z-12, pexigainin acetate, D35, MSI-78A, MG0
[K10E, K11E, F12W-magainin 2], MG2+ [K10E, F12W-magainin-2], MG4+
[F12W-magainin 2], MG6+ [f12W, E19Q-magainin 2 amide], MSI-238,
reversed magainin II analogs [e.g., 53D, 87-ISM, and A87-ISM],
Ala-magainin II amide, magainin II amide), cecropin P1, cecropin A,
cecropin B, indolicidin, nisin, ranalexin, lactoferricin B,
poly-L-lysine, cecropin A (1-8)-magainin II (1-12), cecropin A
(1-8)-melittin (1-12), CA(1-13)-MA(1-13), CA(1-13)-ME(1-13),
gramicidin, gramicidin A, gramicidin D, gramicidin S, alamethicin,
protegrin, histatin, dermaseptin, lentivirus amphipathic peptide or
analog, parasin I, lycotoxin I or II, globomycin, gramicidin S,
surfactin, ralinomycin, valinomycin, polymyxin B, PM2 [(+/-)
1-(4-aminobutyl)-6-benzylindane], PM2c
[(+/-)-6-benzyl-1-(3-carboxypropyl)indane], PM3
[(+/-)1-benzyl-6-(4-aminobutyl)indane], tachyplesin, buforin I or
II, misgurin, melittin, PR-39, PR-26, 9-phenylnonylamine,
(KLAKKLA)n, (KLAKLAK)n, where n=1, 2, or 3, (KALKALK)3, KLGKKLG)n,
and KAAKKAA)n, wherein N=1, 2, or 3, paradaxin, Bac 5, Bac 7,
ceratoxin, mdelin 1 and 5, bombin-like peptides, PGQ, cathelicidin,
HD-5, Oabac5alpha, ChBac5, SMAP-29, Bac7.5, lactoferrin,
granulysin, thionin, hevein and knottin-like peptides, MPG1, 1bAMP,
snakin, lipid transfer proteins, and plant defensins. Exemplary
sequences for the above compounds are provided in Table 1. In some
embodiments, the antimicrobial peptides are synthesized from
L-amino acids, while in other embodiments, the peptides are
synthesized from or comprise D-amino acids.
The compounds listed above can be isolated and purified from
natural sources as appropriate. The compounds may also be produced
recombinantly or synthetically as described below. In some
embodiments, the antimicrobial peptide is included in the media at
a concentration sufficient to lower serum creatinine levels in
kidney transplant recipients as compared to recipients of kidneys
stored without antimicrobial peptides. In other embodiments, the
antimicrobial polypeptide is included in the media at a
concentration sufficient to cause a decrease in delayed graft
function rates of kidneys stored in the media as compared to
unsupplemented media. Preferably, the time for return to baseline
serum creatinine levels is improved by at least 25%, and most
preferably by at least 50%, as compared to control unsupplemented
media. In preferred embodiments, the media of the present invention
comprise one or more antimicrobial polypeptides at a concentration
of about 0.01 to 1000 mg/L. In particularly preferred embodiments,
the media comprises a solution comprising one or more antimicrobial
polypeptides at a concentration of about 0.1 to 5 mg/L.
The present invention is not limited to a particular mechanism of
action. Indeed, an understanding of the mechanism of action is not
necessary to practice the present invention. Nevertheless, the data
summarized in Example 10 demonstrates that the addition of an
antimicrobial polypeptide to standard organ storage solutions
(e.g., UW solution) results in both the stabilization of
cytoskeletal structure and an increased ability of the cytoskeleton
to reassemble upon reperfusion. It is particularly notable that the
antimicrobial polypeptide stabilized both actin filaments and
microtubules.
In some embodiments of the present invention, the antimicrobial
polypeptide is a defensin. In preferred embodiments, the
compositions of the present invention comprise one or more
defensins. In further preferred embodiments, the composition
comprises a solution comprising purified defensins at a
concentration of about 0.01 to 1000 mg/L. In particularly preferred
embodiments, the media comprises a solution comprising defensins at
a concentration of about 0.1 to 5 mg/L. In still further preferred
embodiments, the antimicrobial polypeptide is BNP1 (also known as
bactanecin and bovine dodecapeptide). In certain embodiments, the
defensin comprises the following consensus sequence: (SEQ ID
NO:96-X.sub.1 CN.sub.1 CRN.sub.2 CN.sub.3 ERN.sub.4 CN.sub.5
GN.sub.6 CCX.sub.2, wherein N and X represent conservatively or
nonconservatively substituted amino acids and N.sub.1 =1, N.sub.2
=3 or 4, N.sub.3 =3 or 4, N.sub.4 =1, 2, or 3, N.sub.6 =5-9,
X.sub.1 and X.sub.2 may be present, absent, or equal from 1-2.
The present invention is not limited to any particular defensin.
Indeed, media comprising a variety of defensins are contemplated.
Representative defensins are provided in Tables 1 and 2 below. In
general, defensins are a family of highly cross-linked,
structurally homologous antimicrobial peptides found in the
azurophil granules of polymorphonuclear leukocytes (PMN's) with
homologous peptides being present in macrophages (e.g., Selsted et
al., Infect. Immun. 45:150-154 [1984]). Originally described as
"Lysosomal Cationic Peptides" in rabbit and guinea pig PMN (Zeya et
al., Science 154:1049-1051 [1966]; Zeya et al., J. Exp. Med.
127:927-941 [1968]; Zeya et al., Lab. Invest. 24:229-236 [1971];
Selsted et al., [1984], supra.), this mixture was found to account
for most of the microbicidal activity of the crude rabbit PMN
extract against various microorganisms (Zeya et al., [1966], supra;
Lehrer et al., J. Infect. Dis. 136:96-99 [1977]; Lehrer et al.,
Infect. Immun. 11:1226-1234[1975]). Six rabbit neutrophil defensins
have been individually purified and are designated NP-1, NP-2,
NP-3A, NP-3B, NP-4, and NP-5. Their amino acid sequences were
determined, and their broad spectra of activity were demonstrated
against a number of bacteria (Selsted et al., Infect. Immun.
45:150-154 [1984]), viruses (Lehrer et al., J. Virol. 54:467
[1985]), and fungi (Selsted et al., Infect. Immun. 49:202-206
[1985]; Segal et al, 151:890-894 [1985]). Defensins have also been
shown to possess mitogenic activity (e.g., Murphy et al., J. Cell.
Physiol. 155:408-13 [1993]).
Four peptides of the defensin family have been isolated from human
PMN's and are designated HNP-1, HNP-2, HNP-3, and HNP-4 (Ganz et
al., J. Clin. Invest. 76:1427-1435 [1985]; Wilde et al., J. Biol.
Chem. 264:11200-11203 [1989]). The amino acid sequences of HNP-1,
HNP-2, and HNP-3 differ from each other only in their amino
terminal residues, while each of the human defensins are identical
to the six rabbit peptides in 10 or 11 of their 29 to 30 residues.
These are the same 10 or 11 residues that are shared by all six
rabbit peptides. Human defensin peptides have been shown to share
with the rabbit defensins a broad spectrum of antimicrobial
activity against bacteria, fungi, and enveloped viruses (Ganz et
al., [1985], supra).
Three defensins designated RatNP-1, RatNP-2, and RatNP-4, have been
isolated from rat (Eisenhauer et al., Infection and Immunity
57:2021-2027 [1989]). A guinea pig defensin (GPNP) has also been
isolated, purified, sequenced and its broad spectrum antimicrobial
properties verified (Selsted et al, Infect. Immun. 55:2281-2286
[1987]). Eight of its 31 residues were among those invariant in six
rabbit and three human defensin peptides. The sequence of GPNP also
included three nonconservative substitutions in positions otherwise
invariant in the human and rabbit peptides. Of the defensins tested
in a quantitative assay HNP-1, RatNP-1, and rabbit NP-1 possess the
most potent antimicrobial properties, while NP-5 possesses the
least amount of antimicrobial activity when tested against a panel
of organisms in stationary growth phase (Selsted et al., Infect.
Immun. 45:150-154 [1984]; Ganz et al., J. Clin. Invest.
76:1427-1435 [1985]). Defensin peptides are further described in
U.S. Pat. Nos. 4,543,252; 4,659,692; and 4,705,777 (each of which
is incorporated herein by reference).
Accordingly, in some embodiments, the media comprises one or more
defensins selected from the group consisting of SEQ ID NOs: 37-95.
In particularly preferred embodiments, the media comprises bovine
defensin peptide (BNP-1; SEQ ID NO: 37, Romeo et al., J. Biol.
Chem. 263(15):9573-9575 [1988]). In some embodiments, the defensin
is included in the media at a concentration sufficient to lower
serum creatinine levels in kidney transplant recipients as compared
to recipients of kidneys stored without defensin peptides. Defensin
peptides suitable for use in the methods and compositions of the
present invention include natural defensin peptides isolated from
known cellular sources, synthetic peptides produced by solid phase
or recombinant DNA techniques, and defensin analogs which may be
smaller peptides or other molecules having similar binding and
biological activity as the natural defensin peptides (e.g., peptide
mimetics). Methods for the purification of defensin peptides are
described in U.S. Pat. Nos. 4,543,252; 4,659,692; and 4,705,777,
the disclosures of which are incorporated herein by reference.
In preferred embodiments, suitable synthetic peptides will usually
comprise all or part of the amino acid sequence of a known peptide,
more preferably incorporating at least some of the conserved
regions identified in Table 2. In particularly preferred
embodiments, the synthetic peptides incorporate at least one of the
conserved regions, more usually incorporating two of the conserved
regions, preferably conserving at least three of the conserved
regions, and more preferably conserving four or more of the
conserved regions. In preferred embodiments, the synthetic peptides
comprise fifty amino acids or fewer, although there may be
advantages in increasing the size of the peptide above that of the
natural peptides in certain instances. In certain embodiments, the
peptides have a length in the range from about 10 to 50 amino
acids, preferably being in the range from about 10 to 40 amino
acids, and most preferably being in the range from about 30 to 35
amino acids which corresponds generally to the length of the
natural defensin peptides.
In some cases, it may be desirable to incorporate one or more
non-natural amino acids in the synthetic defensin peptides of the
present invention. In preferred embodiments, non-natural amino
acids comprise at least an N-terminus and a C-terminus and have
side chains that are either identical to or chemically modified or
substituted from a natural amino acid counterpart. An example of a
non-natural amino acid is an optical isomer of a
naturally-occurring L-amino acid, such as a peptide containing all
D-amino acids. Examples of the synthesis of peptides containing all
D-amino acids include Merrifield et al., Ciba Found Symp. 186:5-26
(1994); Wade et al., Proc. Natl. Acad. Sci. USA 87(12):4761-5
(1990); and U.S. Pat. No. 5,792,831, which is herein incorporated
by reference. Examples of chemical modifications or substitutions
include hydroxylation or fluorination of C--H bonds within natural
amino acids. Such techniques are used in the manufacture of drug
analogs of biological compounds and are known to one of ordinary
skill in the art.
Synthetic peptides having biological and binding activity the same
or similar to that of natural defensin peptides may be produced by
either of two exemplary approaches. First, the polypeptides may be
produced by the well-known Merrifield solid-phase chemical
synthesis method wherein amino acids are sequentially added to a
growing chain (Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156
[1963]). Automatic peptide synthesis equipment is available from
several commercial suppliers, including PE Biosystems, Inc., Foster
City, Calif., Beckman Instruments, Inc., Waldwick, N.J.; and
Biosearch, Inc., San Raphael, Calif. Using such automatic
synthesizers according to manufacturer's instructions, peptides may
be produced in gram quantities for use in the present
invention.
Second, the synthetic defensin peptides of the present invention
may be synthesized by recombinant techniques involving the
expression in cultured cells of recombinant DNA molecules encoding
a gene for a desired portion of a natural or analog defensin
molecule. The gene encoding the defensin peptide may itself be
natural or synthetic. Conveniently, polynucleotides may be
synthesized by well known techniques based on the desired amino
acid sequence. For example, short single-stranded DNA fragments may
be prepared by the phosphoramidite method (Beaucage et al., Tetra.
Lett. 22:1859-1862 [1981]). A double-stranded fragment may then be
obtained either by synthesizing the complementary strand and
annealing the strands together under appropriate conditions or by
adding the complementary strand using DNA polymerase under
appropriate conditions or by adding the complementary strand using
DNA polymerase with an appropriate primer sequence. The natural or
synthetic DNA fragments coding for the desired defensin peptide may
then be incorporated in a suitable DNA construct capable of
introduction to and expression in an in vitro cell culture. The DNA
fragments can be portions or variants of wild-type nucleic acids
encoding defensins. Suitable variants include those both with
conservative and nonconservative amino acid substitutions.
The methods and compositions of the present invention may also
employ synthetic non-peptide compositions that have biological
activity functionally comparable to that of the known defensin
peptides. By functionally comparable, it is meant that the shape,
size, flexibility, and electronic configuration of the non-peptide
molecule is such that the biological activity of the molecule is
similar to the defensin peptides. In particular, the non-peptide
molecules should display comparable mitogenic activity and/or
antimicrobial activity or pore forming ability, preferably
possessing both activities. Such non-peptide molecules will
typically be small molecules having a molecular weight in the range
from about 100 to 1000 daltons. The use of such small molecules is
frequently advantageous in the preparation of pharmacological
compositions. Candidate mimetics can be screened in large numbers
to identify those having the desired activity.
The identification of such nonpeptide analog molecules can be
performed using techniques known in the art of drug design. Such
techniques include, but are not limited to, self-consistent field
(SCF) analysis, configuration interaction (CI) analysis, and normal
mode dynamics computer analysis, all of which are well described in
the scientific literature (Rein et al., Computer-Assisted Modeling
of Receptor-Ligand Interactions, Alan Liss, N.Y., [1989]).
Preparation of the identified compounds will depend on the desired
characteristics of the compounds and will involve standard chemical
synthetic techniques (Cary et al., Advanced Organic Chemistry, part
B, Plenum Press, New York [1983]).
TABLE 1 Antimicrobial Peptides SEQ ID NO: Name Organism Sequence 1
lingual antimicrobial Bos taurus
mrlhhlllallflvlsagsgftqgvrnsqscrrnkgicvp peptide precursor
ircpgsmrqigtclgaqvkccrrk (Magainin) 2 antimicrobial peptide Xenopus
laevis gvlsnvigylkklgtgalnavlkq PGQ 3 Xenopsin Xenopus laevis
mykgiflcvllavicanslatpssdadedndeveryvrgw
askigqtlgkiakvglkeliqpkreamlrsaeaqgkrpwil 4 magainin precursor
Xenopus laevis mfkglficsliavicanalpqpeasadedmderevrgigk
flhsagkfgkafvgeimkskrdaeavgpeafadedldere
vrgigkflhsakkfgkafvgeimnskrdaeavgpeafade
dlderevrgigkflhsakkfgkafvgeimnskrdaeavgp
eafadedlderevrgigkflhsakkfgkafvgeimnskrd
aeavgpeafadedfderevrgigkflhsakkfgkafvgei
mnskrdaeavgpeafadedlderevrgigkflhsakkfgk afvgeimnskrdaeavddrrwve 5
tachyplesin I Tachypleus kwcfrvcyrgicyrrcr gigas 6 tachyplesin II
Tachypleus rwcfrvcyrgicyrkcr gigas 7 buforin I Bufo bufo
msgrgkqggkvrakaktrssraglqfpvgrvhrllrkgny gagarizans
aqrvgagapvylaavleyltaeilelagnaardnkktrii
prhlqlavrndeelnkllggvtiaqggvlpniqavllpkt esskpaksk 8 buforin II
Bufo bufo trssraglqfpvgrvhrllrk gagarizans 9 cecropin A Bombyx mori
mnfvrilsfvfalvlalgavsaapeprwklfkkiekvgrn vrdglikagpaiavigqakslgk 10
cecropin B Bombyx mori mnfakilsfvfalvlalsmtsaapeprwkifkkiekmgrn
irdgivkagpaievlgsakaigk 11 cecropin C Drosophila
mnfykifvfvalilaisigqseagwlkklgkrierigqht melanogaster
rdatiqglgiaqqaanvaatarg 12 cecropin P1 Sus scrofa
swlsktakklensakkrisegiaiaiqggpr 13 indolicidin Bos taurus
ilpwkwpwwpwrr 14 nisin Lactococcus
itsislctpgcktgalmgcnmktatchcsihvsk lactis 15 ranalexin Rana
flgglikivpamicavtkkc catesbeiana 16 lactoferricin B Bos taurus
fkcrrwqwrmkklgapsitcvrraf 17 protegrin-1 Sus scrofa
rggrlcycrrrfcvcvgrx 18 protegrin-2 Sus scrofa ggrlcycrrrfcicvg 19
histatin precursor Homo sapiens
mkffvfalilalmlsmtgadshakrhhgykrkfhekhhsh rgyrsnylydn 20 histatin 1
Macaca dsheerhhgrhghhkygrkfhekhhshrgyrsnylydn fascicularis 21
dermaseptin Phyllomedusa alwktmlkklgtmalhagkaalgaaadtisqtq sauvagei
22 dermaseptin 2 Phyllomedusa alwftmlkklgtmalhagkaalgaaantisqgtq
sauvagei 23 dermaseptin 3 Phyllomedusa
alwknmlkgigklagkaalgavkklvgaes sauvagei 24 misgurin Misgurnus
rqrveelskfskkgaaarrrk anguillicaudatus 25 melittin Apis mellifera
gigavlkvlttglpaliswisrkkrqq 26 pardaxin-1 Pardachirus
gffalipkiissplfktllsavgsalsssgeqe pavoninus 27 pardaxin-2
Pardachirus gffalipkiisspifktllsavgsalsssggqe pavoninus 28
bactenecin 5 precursor Bos taurus
metqraslslgrcslwlllllglvlpsasaqalsyreavlr
avdqfnersseanlyrlleldptpnddldpgtrkpvsfrv
ketdcprtsqqpleqcdfkenglvkqcvgtvtldpsndqf
dincnelqsvrfippirrppirppfyppfrppirppifpp irppfrpplgpfpgrr 29
bactenecin precursor Bos taurus
metpraslslgrwslwllllglalpsasaqalsyreavlr
avdqlneqssepniyrlleldqppqddedpdspkrvsfrv
ketvcsrttqqppeqcdfkengllkrcegtvtldqvrgnf
ditcnnhqsiritkqpwappqaarlcrivvirvcr 30 ceratotoxin A Ceratitis
sigsalkkalpvakkigkialpiakaalp capitata 31 ceratotoxin B Ceratitis
sigsafkkalpvakkigkaalpiakaalp capitata 32 cathelicidin
antimicrobial Homo sapiens mktqrnghslgrwslvllllglvmplaiiaqvlsykeavl
peptide raidginqrssdanlyrlldldprptmdgdpdtpkpvsft
vketvcprttqqspedcdfkkdglvkrcmgtvtlnqargs
fdiscdkdnkrfallgdffrkskekigkefkrivqrikdf lrnlvprtes 33 myeloid
cathelicidin 3 Equus caballus
metqrntrclgrwsplllllglvippattqalsykeavlr
avdglnqrssdenlyrlleldplpkgdkdsdtpkpvsfmv
ketvcprimkqtpeqcdfkenglvkqcvgtvildpvkdyf
dascdepqrvkrfhsvgsliqrhqqmirdkseatrhgiri itrpklllas 34 myeloid
antimicrobial Bos taurus metqraslslgrwslwllllglalpsasaqalsyreavlr
peptide BMAP-28 avdqlneksseanlyrlleldpppkeddenpnipkpvsfr
vketvcprtsqqspeqcdfkengllkecvgtvtldqvgsn
fditcavpqsvgglrslgrkilrawkkygpiivpiirig 35 myeloid cathelicidin 1
Equus caballus metqrntrclgrwsplllllglvippattqalsykeavlr
avdglnqrssdenlyrlleldplpkgdkdsdtpkpvsfmv
ketvcprimkqtpeqcdfkenglvkqcvgtvilgpvkdhf
dvscgepqrvkrfgrlaksflrmrillprrkillas 36 SMAP 29 Ovis aries
metqraslslgrcslwllllglalpsasaqvlsyreavlr
aadqlneksseanlyrlleldpppkqddensnipkpvsfr
vketvcprtsqqpaeqcdfkengllkecvgtvfldqvrnn
fditcaepqsvrglrrlgrkiahgvkkygptvlriiriag 37 BNP-1 Bos taurus
rlcrivvirvcr 38 HNP-1 Homo sapiens acycripaciagerrygtciyqgrlwafcc
39 HNP-2 Homo sapiens cycripaciagerrygtciyqgrlwafcc 40 HNP-3 Homo
sapiens dcycripaciagerrygtciyqgrlwafcc 41 HNP-4 Homo sapiens
vcscrlvfcrrtelrvgncliggvsftycctrv 42 NP-1 Oryctolagus
vvcacrralclprerragfcrirgrihplccrr cuniculus 43 NP-2 Oryctolagus
vvcacrralclplerragfcrirgrihplccrr cuniculus 44 NP-3A Oryctolagus
gicacrrrfcpnserfsgycrvngaryvrccsrr cuniculus 45 NP-3B Oryctolagus
grcvcrkqllcsyrerrigdckirgvrfpfccpr cuniculus 46 NP-4 Oryctolagus
vsctcrrfscgfgerasgsctvnggvrhtlccrr cuniculus 47 NP-5 Oryctolagus
vfctcrgflcgsgerasgsctingvrhtlccrr cuniculus 48 RatNP-1 Rattus
vtcycrrtrcgfrerlsgacgyrgriyrlccr norvegicus 49 Rat-NP-3 Rattus
cscrysscrfgerllsgacrlngriyrlcc norvegicus 50 Rat-NP-4 Rattus
actcrigacvsgerltgacglngriyrlccr norvegicus 51 GPNP Guinea pig
rrcicttrtcrfpyrrlgtcifqnrvytfcc 52 beta defensin-3 Homo sapiens
mrihyllfallflflvpvpghggiintlqkyycrvrggrc
avlsclpkeeqigkcstrgrkccrrkk 53 theta defensin-1 Macaca
rcictrgfcrclcrrgvc mulatta 54 defensin CUA1 Helianthus
mkssmkmfaalllvvmcllanemggplvveartcesqshk annuus
fkgtclsdtncanvchserfsggkcrgfrrrcfctthc 55 defensin SD2 Helianthus
mkssmkmfaalllvvmcllanemggplvveartcesqshk annuus
fkgtclsdtncanvchserfsggkcrgfrrrcfctthc 56 neutrophil defensin 2
Macaca acycripaclagerrygtcfymgrvwafcc mulatta 57 4 KDA defensin
Androctonus gfgcpfnqgachrhcrsirrrggycaglfkqtctcyr australis hector
58 defensin Mytilus gfgcpnnyqchrhcksipgrcggycggxhrlrctcyrc
galloprovincialis 59 defensin AMP1 Heuchera
dgvklcdvpsgtwsghcgssskcsqqckdrehfayggach sanguinea yqfpsvkcfckrqc
60 defensin AMP1 Clitoria nlcerasltwtgncgntghcdtqcrnwesakhgachkrgn
ternatea wkcfcyfnc 61 cysteine-rich cryptdin-1 Mus musculus
mkklvllfalvllafqvqadsiqntdeetkteeqpgekdq homolog
avsvsfgdpqgsalqdaalgwgrrcpqcprcpscpscprc prcprckcnpk 62
beta-defensin-9 Bos taurus qgvrnfvtcrinrgfcvpircpghrrqigtclgpqikccr
63 beta-defensin-7 Bos taurus
qgvrnfvtcrinrgfcvpircpghrrqigtclgprikccr 64 beta-defensin-6 Bos
taurus qgvrnhvtcriyggfcvpircpgrtrqigtcfgrpvkccrrw 65
beta-defensin-5 Bos taurus qvvrnpqscrwnmgvcipiscpgnmrqigtcfgprvpccr
66 beta-defensin-4 Bos taurus
qrvrnpqscrwnmgvcipflcrvgmrqigtcfgprvpccrr 67 beta-defensin-3 Bos
taurus qgvrnhvtcrinrgfcvpircpgrtrqigtcfgprikccrsw 68
beta-defensin-10 Bos taurus
qgvrsylscwgnrgicllnrcpgrmrqigtclaprvkccr 69 beta-defensin-13 Bos
taurus sgisgplscgrnggvcipircpvpmrqigtcfgrpvkccrsw 70
beta-defensin-1 Bos taurus dfaschtnggiclpnrcpghmiqigicfrprvkccrsw
71 coleoptericin Zophobas
slqggapnfpqpsqqnggwqvspdlgrddkgntrgqieiq atratus
nkgkdhdfnagwgkvirgpnkakptwhvggtyrr 72 beta defensin-3 Homo sapiens
mrihyllfallflflvpvpghggiintlqkyycrvrggrc
avlsclpkeeqigkcstrgrkccrrkk 73 defensin C Aedes aegypti
atcdllsgfgvgdsacaahciargnrggycnskkvcvcrn 74 defensin B Mytilus
edulis gfgcpndypchrhcksipgryggycggxhrlrctc 75 sapecin C Sarcophaga
atcdllsgigvqhsacalhcvfrgnrggyctgkgicvcrn peregrina 76 macrophage
antibiotic Oryctolagus mrtlallaaillvalqaqaehvsvsidevvdqqppqaedq
peptide MCP-1 cuniculus dvaiyvkehessalealgvkagvvcacrralclprerrag
fcrirgrihplccrr 77 cryptdin-2 Mus musculus
mkplvllsalvllsfqvqadpiqntdeetkteeqsgeedq
avsvsfgdregaslqeeslrdlvcycrtrgckrrermngt crkghlmytlcc 78 cryptdin-5
Mus musculus mktfvllsalvllafqvqadpihktdeetnteeqpgeedq
avsisfggqegsalheelskklicycrirgckrrervfgt crnlfltfvfccs 79 cryptdin
12 Mus musculus lrdlvcycrargckgrermngtcrkghllymlccr 80 defensin
Pyrrhocoris atcdilsfqsqwvtpnhagcalhcvikgykggqckitvchcrr apterus 81
defensin R-5 Rattus vtcycrstrcgfrerlsgacgyrgriyrlccr norvegicus 82
defensin R-2 Rattus vtcscrtsscrfgerlsgacrlngriyrlcc norvegicus 83
defensin NP-6 Oryctolagus gicacrrrfclnfeqfsgycrvngaryvrccsrr
cuniculus 84 beta-defensin-2 Pan
mrvlyllfsflfiflmplpgvfggisdpvtclksgaichp troglodytes
vfcprrykqigtcglpgtkcckkp 85 beta-defensin-2 Homo sapiens
mrvlyllfsflfiflmplpgvfggigdpvtclksgaichp vfcprrykqigtcglpgtkcckkp
86 beta-defensin-1 Homo sapiens
mrtsylllftlclllsemasggnfltglghrsdhyncvss
ggqclysacpiftkiqgtcyrgkakcck 87 beta-defensin-1 Capra hircus
mrlhhlllvlfflvlsagsgftqgirsrrschrnkgvcal trcprnmrqigtcfgppvkccrkk
88 beta defensin-2 Capra hircus
mrlhhlllalfflvlsagsgftqgiinhrscyrnkgvcap arcprnmrqigtchgppvkccrkk
89 defensin-3 Macaca mrtlvilaaillvalqaqaeplqartdeataaqeqiptdn
mulatta pevvvslawdeslapkdsvpglrknmacycripaclager rygtcfyrrrvwafcc
90 defensin-1 Macaca mrtlvilaaillvalqaqaeplqartdeataaqeqiptdn
mulatta pevvvslawdeslapkdsvpglrknmacycripaclager rygtcfylgrvwafcc
91 neutrophil defensin 1 Mesocricetus
vtcfcrrrgcasrerhigycrfgntiyrlccrr auratus 92 neutrophil defensin 1
Mesocricetus cfckrpvcdsgetqigycrlgntfyrlccrq auratus 93 Gallinacin
1-alpha Gallus gallus grksdcfrkngfcaflkcpyltlisgkcsrfhlcckriw 94
defensin Allomyrina vtcdllsfeakgfaanhslcaahclaigrrggscergvcicrr
dichotoma 95 neutrophil cationic Cavia
rrcicttrtcrfpyrrlgtcifqnrvytfcc peptide 1 porcellus
TABLE 2 Defensins SEQ ID NO Name Organism Sequence 38 HNP-1 Human
ACYCRIPACIAGERRYGTCIYQGRLWAFCC 39 HNP-2 Human
CYCRIPACIAGERRYGTCIYQGRLWAFCC 40 HNP-3 Human
DCYCRIPACIAGERRYGTCIYQGRLWAFCC 41 HNP-4 Human
VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTR V 42 NP-1 Rabbit
VVCACRRALCLPRERRAGFCRIRGRIHPLCCRR 43 NP-2 Rabbit
VVCACRRALCLPLERRAGFCRIRGRIHPLCCRR 44 NP-3A Rabbit
GICACRRRFCPNSERFSGYCRVNGARYVRCCS RR 45 NP-3B Rabbit
GRCVCRKQLLCSYRERRIGDCKIRGVRFPFCCP R 46 NP-4 Rabbit
VSCTCRRFSCGFGERASGSCTVNGVRHTLCCR R 47 NP-5 Rabbit
VFCTCRGFLCGSGERASGSCTINGVRHTLCCR R 48 RatNP-1 Rat
VTCYCRRTRCGFRERLSGACGYRGRIYRLCCR 49 Rat-NP-3 Rat
CSCRYSSCRFGERLLSGACRLNGRIYRLCC 50 Rat-NP-4 Rat
ACTCRIGACVSGERLTGACGLNGRIYRLCCR 51 GPNP Guinea pig
RRCICTTRTCRFPYRRLGTCIFQNRVYTFCC
B. Cell Surface Receptor Binding Compounds
In some embodiments of the present invention, media for preserving
organs comprise one or more cell surface receptor binding
compounds. Cell surface receptor binding compounds useful in the
present invention include, but are not limited to, the following
broad classes of cytoactive compounds: Insulin, Insulin like Growth
Factors such as IGF-I, IGF-II, and IGF-BP; Epidermal Growth Factors
such as .alpha.-EGF and .beta.-EGF; EGF-like molecules such as
Keratinocyte-derived growth factor (which is identical to KAF,
KDGF, and amphiregulin) and vaccinia virus growth factor (VVGF);
Fibroblast Growth Factors such as FGF-1 (Basic FGF Protein), FGF-2
(Acidic FGF Protein), FGF-3 (Int-2), FGF-4 (Hst-1), FGF-5, FGF-6,
and FGF-7 (identical to KGF); FGF-Related Growth Factors such as
Endothelial Cell Growth Factors (e.g., ECGF-.alpha. and
ECGF-.beta.); FGF- and ECGF-Related Growth Factors such as
Endothelial cell stimulating angiogenesis factor and Tumor
angiogenesis factor, Retina-Derived Growth Factor (RDGF), Vascular
endothelium growth factor (VEGF), Brain-Derived Growth Factor (BDGF
A- and -B), Astroglial Growth Factors (AGF 1 and 2),
Omentum-derived factor (ODF), Fibroblast-Stimulating factor (FSF),
and Embryonal Carcinoma-Derived Growth Factor; Neurotrophic Growth
Factors such as .alpha.-NGF, .beta.-NGF, .gamma.-NGF, Brain-Derived
Neurotrophic Factor (BDNF), Neurotrophin-3, Neurotrophin-4, and
Ciliary Nuerotrophic Factor (CNTF); Glial Growth Factors such as
GGF-I, GGF-II, GGF-III, Glia Maturation Factor (GMF), and
Glial-Derived Nuerotrophic Factor (GDNF); Organ-Specific Growth
Factors such as Liver Growth Factors (e.g., Hepatopoietin A,
Hepatopoietin B, and Hepatocyte Growth Factors (HCGF or HGF),
Prostate Growth Factors (e.g., Prostate-Derived Growth Factors
[PGF] and Bone Marrow-Derived Prostate Growth Factor), Mammary
Growth Factors (e.g., Mammary-Derived Growth Factor 1 [MDGF-1] and
Mammary Tumor-Derived Factor [MTGF]), and Heart Growth Factors
(e.g., Nonmyocyte-Derived Growth Factor [NMDGF]); Cell-Specific
Growth Factors such as Melanocyte Growth Factors (e.g.,
Melanocyte-Stimulating Hormone [.alpha.-, .beta.-, and .gamma.-MSH]
and Melanoma Growth-Stimulating Activity [MGSA]), Angiogenic
Factors (e.g., Angiogenin, Angiotropin, Platelet-Derived ECGF,
VEGF, and Pleiotrophin), Transforming Growth Factors (e.g.,
TGF-.alpha., TGF-.beta., and TGF-like Growth Factors such as
TGF-.beta..sub.2, TGF-.beta..sub.3, TGF-e, GDF-1, CDGF and
Tumor-Derived TGF-.beta.-like Factors), ND-TGF, and Human
epithelial transforming factor [h-TGFe]); Regulatory Peptides with
Growth Factor-like Properties such as Bombesin and Bombesin-like
peptides (e.g., Ranatensin, and Litorin], Angiotensin, Endothelin,
Atrial Natriuretic Factor, Vasoactive Intestinal Peptide, and
Bradykinin; Cytokines such as the interleukins IL-1 (e.g.,
Osteoclast-activating factor [OAF], Lymphocyte-activating factor
[LAF], Hepatocyte-stimulating factor [HSF], Fibroblast-activating
factor [FAF], B-cell-activating factor [BAF], Tumor inhibitory
factor 2 [TIF-2], Keratinocyte-derived T-cell growth factor
[KD-TCGF]), IL-2 (T-cell growth factor [TCGF], T-cell mitogenic
factor [TCMF]), IL-3 (e.g., Hematopoietin, Multipotential
colony-stimulating factor [multi-CSF], Multilineage
colony-stimulating activity [multi-CSA], Mast cell growth factor
[MCGF], Erythroid burst-promoting activity [BPA-E], IL-4 (e.g.,
B-cell growth factor I [BCGF-I], B-cell stimulatory factor 1
[BSF-1]), IL-5 (e.g., B-cell growth factor II [BCGF-II], Eosinophil
colony-stimulating factor [Eo-CSF], Immunoglobulin A-enhancing
factor [IgA-EF], T-cell replacing factor [TCRF]), IL-6 (B-cell
stimulatory factor 2 [BSF-2], B-cell hybridoma growth factor
[BCHGF], Interferon P2 [IFN-B], T-cell activating factor [TAF],
IL-7 (e.g., Lymphopoietin 1 [LP-1], Pre-B-cell growth factor
[pre-BCGF]), IL-8 (Monocyte-derived neutrophil chemotactic factor
[MDNCF], Granulocyte chemotatic factor [GCF], Neutrophil-activating
peptide 1 [NAP-1], Leukocyte adhesion inhibitor [LAI], T-lymphocyte
chemotactic factor [TLCF]), IL-9 (e.g., T-cell growth factor III
[TCGF-III], Factor P40, MegaKaryoblast growth factor (MKBGF), Mast
cell growth enhancing activity [MEA or MCGEA]), IL-10 (e.g.,
Cytokine synthesis inhibitory factor [CSIF]), IL-11 (e.g., Stromal
cell-derived cytokine [SCDC]), IL-12 (e.g., Natural killer cell
stimulating factor [NKCSF or NKSF], Cytotoxic lymphocyte maturation
factor [CLMF]), TNF-.alpha. (Cachectin), TNF-.beta. (Lymphotoxin),
LIF (Differentiation-inducing factor [DIF],
Differentiation-inducing activity [DIA], D factor, Human
interleukin for DA cells [HILDA], Hepatocyte stimulating factor III
[HSF-III], Cholinergic neuronal differentiation factor [CNDF],
CSF-1 (Macrophage colony-stimulating factor [M-CSF]), CSF-2
(Granulocyte-macrophage colony-stimulating factor [GM-CSF]), CSF-3
(Granulocyte colony-stimulating factor [G-CSF]), and
erythropoietin; Platelet-derived growth factors (e.g., PDGF-A,
PDGF-B, PDGF-AB, p28-sis, and p26-cis), and Bone Morphogenetic
protein (BMP), neuropeptides (e.g., Substance P, calcitonin
gene-regulated peptide, and neuropeptide Y), and neurotransmitters
(e.g., norepinephrine and acetylcholine).
In some preferred embodiments, EGF, IGF-1, and/or NGF are included
in the media at a concentration of about 1 ng/ml to 100 ng/ml, most
preferably about 10 ng/ml. In other preferred embodiments,
substance P is included at a concentration of about 0.1 .mu.g/ml to
100 .mu.g/ml, most preferably about 2.5 .mu.g/ml. In some
embodiments, NGF is deleted as it may not be essential for
suppressing delayed graft function. In some embodiments, the cell
surface receptor binding compound is included in the media at a
concentration sufficient to lower serum creatinine levels in kidney
transplant recipients as compared to recipients of kidneys stored
without cell surface receptor binding compounds. In other
embodiments, the cell surface receptor binding compound(s) are
included in the media at concentrations sufficient to cause a
decrease in delayed graft function rates of kidneys stored in the
media as compared to unsupplemented media. Preferably, the time for
return to baseline serum creatinine levels is improved by at least
25%, and most preferably by at least 50%, as compared to control
unsupplemented media.
Suitable cell surface receptor binding compounds may be obtained
from commercial sources, purified from natural sources, or be
produced by recombinant methods. Recombinant cell surface receptor
binding compounds can be produced from wild-type coding sequences
or from variant sequences that encode functional cell surface
receptor binding compounds. Suitable cell surface receptor binding
compounds also include analogs which may be smaller peptides or
other molecules having similar binding and biological activity as
the natural cell surface receptor binding compounds. Methods for
producing cell surface receptor binding compounds are described in
U.S. Pat. Nos. 5,183,805; 5,218,093; 5,130,298; 5,639,664;
5,457,034; 5,210,185; 5,470828; 5,650,496; 5,998,376; and
5,410,019; all of which are incorporated herein by reference.
C. Other Transplant Media Components
In certain embodiments, a number of other components are utilized
in the media of the present invention to provide the proper balance
of electrolytes, a physiological pH, proper oncotic pressure, etc.
Therefore, it is contemplated that the media comprises one or more
components selected from one or more of the following general
groups: 1) electrolytes; 2) oncotic agents; 3) buffers; 4) energy
sources; 5) impermeant anions; 6) free radical scavengers; and/or
7) ATP sources. Examples of these components are provided below
along with several exemplary media formulations. Examples of media
that can be supplemented with defensins include VIASPAN (U.S. Pat.
Nos. 4,798,824; 4,873,230; and 5,696,152, each of which is
incorporated herein by reference) and various HYPOTHERMOSOL
formulations (U.S. Pat. Nos. 5,514,536 and 6,045,990, each of which
is incorporated herein by reference).
1) Electrolytes
In some embodiments of the present invention, the media comprises
electrolytes (e.g., sodium, potassium, calcium, magnesium,
chloride, sulfate, bicarbonate, and phosphate) in concentrations
approximating those found in blood plasma. For example, in some
embodiments, potassium and phosphate are provided as KH.sub.2
PO.sub.4 in range from about 10 to 50 mM, preferably about 25 mM;
magnesium is provided as magnesium gluconate in a range of from
about 1 to 10 mM, preferably about 5 mM; sodium is provided as
sodium gluconate in a range of from about 50 mM to about 150 mM,
preferably about 105 mM; and calcium and chloride are provided as
CaCl.sub.2 in a range of from about 0.1 to 5.0 mM, preferably about
0.5 mM.
In other embodiments, the concentration of individual electrolytes
may be varied from physiological concentrations. For example, it is
known that membrane pumps of cells are turned off during
hypothermia. As a result, potassium and sodium exchange passively
across the cell membrane. The media can be adjusted to compensate
for the influx of sodium and efflux of potassium by providing
potassium in a range of from about 35 to 45 mM and sodium in a
range of from about 80 to 120 mM. In further embodiments of the
present invention, divalent cations can be included in an amount
sufficient to displace or block the effect of calcium ions at the
cellular membrane. Accordingly, in some embodiments, Ca.sup.++ is
provided in a range of from about 0.01 mM to 0.1 mM, preferably
from about 0.01 to 0.07 mM, and Mg.sup.++ is provided in a range of
from about 1 mM to 10 mM, preferably about 2.5 mM to 7.5 mM.
2) Oncotic Agents
In some embodiments of the present invention, the media comprises
one or more oncotic agents. In preferred embodiments, the oncotic
agent is included in an amount sufficient to maintain oncotic
pressure equivalent to that of blood plasma. The present invention
is not limited to any particular oncotic agent. Indeed, any oncotic
agent can be used that is of a size that does not readily escape
the circulation by traversing the fenestrations of the capillary
bed. Examples of oncotic agents include, but are not limited to,
hydroxyethyl starch, cyclodextrins, and dextran (e.g., Dextran 30,
40, or 50). In preferred embodiments, the media comprises from
about 1% to 10% of the oncotic agent. In particularly preferred
embodiments, the media comprises about 5% of the oncotic agent.
Surprisingly, it has been found that the hydroxyethyl starch
component of VIASPAN can be deleted and good results still
obtained.
3) Buffers
In some embodiments of the present invention, the media comprises
at least one buffer. In preferred embodiments, the concentration of
buffer(s) is sufficient to maintain the pH of the media at a range
of from about 7.0 to 8.0 at 10.degree. C., preferably from about
7.4 to 7.8. The present invention is not limited to the use of any
particular buffer. Indeed, the use of a variety of synthetic and
other buffers is contemplated. Examples of suitable buffers
include, but are not limited to,
N-2-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid
(HEPES), 3-(N-morpholino) propanesulfonic acid (MOPS),
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
2-((2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino) ethanesulfonic
acid (TES), 3-(N-tris(hydroxy-methyl)
methylamino)-2-hydroxypropanesulfonic acid (TAPSO),
4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), pH
range 7.3-8.7, and tris(hydroxymethyl)aminomethane (THAM),
HCO.sub.3, and H.sub.2 PO.sub.4.
4) Energy Sources
In some embodiments of the present invention, the media further
comprises one or more energy or nutrition sources. Examples of
energy sources include, but are not limited to, sucrose, fructose,
glucose, and dextran. Preferably, the concentration of the energy
source is from about 1 mM to 20 mM, most preferably about 10
mM.
5) Impermeant Anions
In some embodiments of the present invention, the media comprises
one or more impermeant anions. The impermeant anion is included to
counteract swelling during cold exposure. The present invention is
not limited to any particular impermeant anion. Indeed, a variety
of impermeant anions are contemplated, including, but not limited
to, gluconate and lactobionate. Preferably, the concentration of
the impermeant anion is from about 50 to 150 mM, most preferably
about 100 mM.
6) Free Radical Scavengers
In some embodiments of the present invention, the media comprises a
free radical scavenger. The present invention is not limited to any
particular free radical scavenger. Indeed, a variety of free
radical scavengers are contemplated including, but not limited to,
mannitol and glutathione. Preferably, the concentration of the free
radical scavenger is from about 1 mM to 10 mM, most preferably
about 3 mM.
7) ATP Substrate
In some embodiments of the present invention, the media comprises
one or more ATP substrates for the regeneration of ATP during
rewarming. The present invention is not limited to any particular
ATP substrate. Indeed, a variety of ATP substrates are
contemplated, including, but not limited to, adenosine, fructose,
adenine, and ribose. Preferably, the concentration of the ATP
substrate is from about 1 mM to 10 mM, most preferably about 5
mM.
8) Osmotic Agents
In some embodiments of the present invention, the media comprises
one or more osmotic agents. Examples of osmotic agents include, but
are not limited to, trehalose (.alpha.-.alpha.-trehalose
dihydrate), raffinose, sucrose and mannitol. In preferred
embodiments, the osmotic agent is provided at a concentration of
about 1 mM to 100 mM, most preferably about 30 mM. In other
embodiments, it is contemplated that trehalose is included in the
media as protectant. Accordingly, in some embodiments, the media
comprises trehalose at a concentration of about 1 mM to 30 mM,
preferably about 20 mM. In other embodiments, trehalose is included
in the media at a concentration sufficient to lower serum
creatinine levels in kidney transplant recipients as compared to
recipients of kidneys stored without antimicrobial peptides.
9) Other Components
In some embodiments of the present invention, the media may further
comprise a variety of additional components. For example, in some
embodiments, the media comprises an inhibitor of xanthine oxidase
(e.g., allopurinol at a concentration of about 0.1 mM to 5 mM, most
preferably about 1 mM). In still further embodiments, the media
comprises an iron-chelating agent (e.g., deferoxamine at a
concentration of from about 0.05 mM to 5 mM, most preferably about
1.0 mM). In additional embodiments, the media comprises a steroidal
agent (e.g., dexamethasone at a concentration of about 1 to 30
mg/liter, most preferably about 16 mg/liter). In other embodiments,
the media comprises hydrocortisone (e.g., at a concentration of
from about 10 ng/ml to 100 ng/ml, preferably about 36 ng/ml). In
still other embodiments, the media comprises ITS (insulin [5
.mu.g/ml], transferrin [5 .mu.g/ml], and selenium [5 ng/ml]). In
some embodiments, the media comprises vitamin C (e.g., at about
1.times.10.sup.-7 M). In other embodiments, the media comprises
protease inhibitors (e.g., phosphoramidon [5 .mu.M], thiorphan [1
.mu.M], bacetracin [1 .mu.M], and encaptopril [5 .mu.M]).
Additionally, the media of the present invention may comprise
additional cytoskeleton stabilizing agents. In particular, agents
such as taxol, discodermolide, epothilone A and B, vinblastine, and
vinchristine may be utilized in some embodiments of the present
invention, in combination with either the antimicrobial
polypeptides or cell surface receptor binding compounds or both.
The use of taxol with UW solution is described in U.S. Pat. No.
5,696,152, incorporated herein by reference.
10) Exemplary Media Formulations
It is contemplated that antimicrobial peptides, other pore forming
agents, and/or cell surface receptor binding compounds can be added
to a variety of media formulations currently being used for organ
preservation and/or other surgical procedures such as cardioplegia.
Non-limiting examples of these media are provided in the Tables
below. It will be recognized that the media may comprise one or
more antimicrobial polypeptides (e.g., a defensin such as BNP-1).
The media described below may also comprise one or more trophic
factors and/or cell surface receptor binding compounds as described
above. Accordingly, in some preferred embodiments, the media is
supplemented with one or more of the following trophic factors:
trehalose (Sigma, St. Louis Mo.; e.g., about 15 mM), substance P
(Sigma; e.g., about 10 .mu.g/ml), IGF-1 (Collaborative Biologicals;
e.g., about 10 ng/ml), EGF (Sigma; e.g., about 10 ng/ml), and NGF
(Sigma [murine] or Genentech [human]; e.g., about 200 ng/ml). In
some preferred embodiments, the transplant media is also
supplemented with dexamethasone (1-20 mg/l, preferably 8 mg/1),
penicillin (20,000-500,000 units, preferably 200,000 units), and
insulin (1-200 units, preferably 40 units) prior to use. In some
embodiments, an antimicrobial polypeptide is not included in the
medium. In some embodiments, the antimicrobial polypeptide and/or
cell surface receptor binding compounds are included in the media
at concentrations sufficient to lower serum creatinine levels in
kidney transplant recipients as compared to recipients of kidneys
stored in control unsupplemented media. In other embodiments, the
antimicrobial polypeptide and/or cell surface receptor binding
compounds are included in the media at concentrations sufficient to
cause a decrease in delayed graft function rates of kidneys stored
in the media as compared to control unsupplemented media.
Preferably, the time for return to baseline serum creatinine levels
is improved by at least 25%, and most preferably by at least 50%,
as compared to control unsupplemented media.
It is contemplated that the media can be provided in a
pre-formulated form (which can be in kit format with instructions,
etc.) which comprises the antimicrobial polypeptide and/or one or
more trophic factors or as a kit comprising at least one container
of base medium (e.g., UW solution (VIASPAN), HTK Solution,
EuroCollins Solution, or Collins Solution)) and a separate
container or containers containing at least one of the
antimicrobial polypeptides and/or one or more cell surface receptor
binding compounds. Therefore, it will be recognized that the Tables
below provide formulations for exemplary supplemented media (i.e.,
the formula of the media after addition of the antimicrobial
polypeptide and at least one cell surface receptor binding
compound) and that the media can be provided in either a
pre-formulated form or supplemented immediately prior to use. In
preferred embodiments, the antimicrobial polypeptide and/or one or
more cell surface receptor binding compounds are provided in stable
form that can be reconstituted. Methods for stabilization include
lyophilization. In embodiments where the antimicrobial polypeptide
and/or one or more cell surface receptor binding compounds are
provided in lyophilized form, they can conveniently reconstituted
prior to use in sterile water or in an aliquot of base medium
(e.g., UW solution) prior to addition to the base medium (e.g., UW
solution). In some embodiments, the kits include instructions for
reconstitution of the antimicrobial polypeptide and/or one or more
cell surface receptor binding compounds and/or for the use of the
supplemented medium for cold storage or machine perfusion of an
organ.
Alternatively, the at least one microbial polypeptide and/or one or
more cell surface receptor binding compounds can be provided as a
separate composition (i.e., a "bullet") that is added to a base
medium. In preferred embodiments, the bullet contains a defensin
and/or one or more of the cell surface receptor binding compounds
described above. In some embodiments, the bullet contains a
defensin and/or one or more of the cell surface receptor binding
compounds above in concentrations that provide the appropriate
concentration when added to one liter, two liters, or five liters
of the base medium. For example, in some preferred embodiments, a
bullet for addition to 1 liter of base medium comprises 1 mg of an
antimicrobial polypeptide (e.g., BNP-1), 10 mg Substance P, 10
.mu.g IGF-1, 10 .mu.g EGF, 200 .mu.g NGF, and an amount of
trehalose sufficient to provide a concentration of 15 mM. In other
preferred embodiments, a bullet for addition to 1 liter of base
medium comprises 1 mg of an antimicrobial polypeptide (e.g.,
BNP-1), 10 mg Substance P, 10 .mu.g IGF-1, and 10 .mu.g EGF. In
still other preferred embodiments, the antimicrobial polypeptide
and/or one or more cell surface receptor binding compounds are
provided in amounts such when the bullet is added to a base
transplant medium and the supplemented medium is used for kidney
storage prior to transplantation, subjects receiving the kidneys
stored in the supplemented medium exhibit a faster return to
baseline serum creatinine levels than patients receiving kidneys
stored in unsupplemented medium.
TABLE 3 Supplemented UW Solution (VIASPAN) Lactobionic acid 100 mM
KOH 100 mM NaOH 20 mM Adenosine 5 mM Allopurinol 1 mM Potassium
Phosphate (Monobasic) 25 mM MgSO.sub.4 5 mM Raffinose 30 mM
Glutathione 3 mM Hydroxyethyl starch 50 g/L Defensin 1 mg/L
dexamethasone 8 mg/L penicillin 200,000 units insulin 40 units pH
7.4
TABLE 4 Supplemented UW Machine Perfusion Solution Hydroxyethyl
starch 50 g/L Potassium gluconate 10 mM Sodium gluconate 90 mM
Potassium Phosphate (Monobasic) 15 mM Glucose 10 mM Glutathione 3
mM HEPES 10 mM Magnesium gluconate 5 mM Calcium chloride 0.5 mM
Ribose 5 mM Adenosine 5 mM Adenine 5 mM Allopurinol 1 mM Mannitol
14 mM Defensin 1 mg/L pH 7.4 Osmolarity 310
TABLE 5 Hypertonic Citrate Solution Na.sup.+ 80 mM K.sup.+ 80 mM
Mg.sup.++ 35 mM Citrate.sup.- 55 mM SO.sub.4.sup.- 35 mM Mannitol
136 mM Defensin 1 mg/L pH 7.1 Osmolarity 400
TABLE 6 HTK Solution Na.sup.+ 15 mM K.sup.+ 10 mM Mg.sup.++ 4 mM
Cb.sup.- 50 mM Tryptophan 2 mM 2-oxoglutarate 1 mM Mannitol 30 mM
Histidine 0.18 mM Histidine HCl 18 mM pH 7.3 Defensin 1 mg/L
Osmolarity 310
TABLE 7 HTK Solution of Bretshneider Ketoglutaric acid 1 mM
Tryptophan 2 mM MgCl.sub.2 4 mM KCl 10 mM NaCl 15 mM Histidine 200
mM Defensin 1 mg/L pH 7.3
TABLE 8 Phosphate Buffered Sucrose Sodium Phosphate Dibasic 53.6 mM
Sodium Phosphate Monobasic 15.5 mM Sucrose 140 mM Defensin 1 mg/L
pH 7.2
TABLE 9 EuroCollins Solution NaHCO.sub.3 10 mM KCl 15 mM K.sub.2
HPO.sub.4 42.5 mM KH.sub.2 PO.sub.4 15.1 mM Glucose 195 mM Defensin
1 mg/L
TABLE 10 Collins C2 Solution K.sub.2 HPO.sub.4 42.5 mM KH.sub.2
PO.sub.4 15.1 mM KCl 15 mM NaHCO.sub.3 10 mM Glucose 140 mM
MgSO.sub.4 30 mM Defensin 1 mg/L
TABLE 11 Supplemented UW Solution (VIASPAN) Lactobionic acid
(potassium lactobionate) 100 mM KOH 100 mM NaOH 20 mM Adenosine 5
mM Allopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM
MgSO.sub.4 5 mM Raffinose 30 mM Glutathione 3 mM Hydroxyethyl
starch 50 g/L BNP-1 1 mg/L Trehalose 15 mM Substance P 10 .mu.g/ml
IGF-1 10 ng/ml EGF 10 ng/ml NGF 200 ng/ml dexamethasone 8 mg/l
penicillin 200,000 units insulin 40 units pH 7.4
TABLE 12 Supplemented UW Solution (VIASPAN) Lactobionic acid
(potassium lactobionate) 100 mM KOH 100 mM NaOH 20 mM Adenosine 5
mM Allopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM
MgSO.sub.4 5 mM Raffinose 30 mM Glutathione 3 mM Hydroxyethyl
starch 50 g/L BNP-1 1 mg/L Substance P 10 .mu.g/ml IGF-1 10 ng/ml
EGF 10 ng/ml dexamethasone 8 mg/l penicillin 200,000 units insulin
40 units pH 7.4
TABLE 13 EuroCollins Solution NaHCO.sub.3 10 mM KCl 15 mM K.sub.2
HPO.sub.4 42.5 mM KH.sub.2 PO.sub.4 15.1 mM Glucose 195 mM
Trehalose 15 mM Substance P 10 .mu.g/ml IGF-1 10 ng/ml EGF 10 ng/ml
NGF 200 ng/ml BNP-1 1 mg/L
TABLE 13 EuroCollins Solution NaHCO.sub.3 10 mM KCl 15 mM K.sub.2
HPO.sub.4 42.5 mM KH.sub.2 PO.sub.4 15.1 mM Glucose 195 mM
Substance P 10 .mu.g/ml IGF-1 10 ng/ml EGF 10 ng/ml BNP-1 1
mg/L
TABLE 14 Supplemented UW Solution (VIASPAN) Lactobionic acid
(potassium lactobionate) 100 mM KOH 100 mM NaOH 20 mM Adenosine 5
mM Allopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM
MgSO.sub.4 5 mM Raffinose 30 mM Glutathione 3 mM Hydroxyethyl
starch 50 g/L Trehalose 15 mM Substance P 10 .mu.g/ml IGF-1 10
ng/ml EGF 10 ng/ml NGF 200 ng/ml dexamethasone 8 mg/l penicillin
200,000 units insulin 40 units pH 7.4
TABLE 15 Supplemented UW Solution (VIASPAN) Lactobionic acid
(potassium lactobionate) 100 mM KOH 100 mM NaOH 20 mM Adenosine 5
mM Allopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM
MgSO.sub.4 5 mM Raffinose 30 mM Glutathione 3 mM Hydroxyethyl
starch 50 g/L Substance P 10 .mu.g/ml IGF-1 10 ng/ml EGF 10 ng/ml
dexamethasone 8 mg/l penicillin 200,000 units insulin 40 units pH
7.4
TABLE 16 EuroCollins Solution NaHCO.sub.3 10 mM KCl 15 mM K.sub.2
HPO.sub.4 42.5 mM KH.sub.2 PO.sub.4 15.1 mM Glucose 195 mM
Trehalose 15 mM Substance P 10 .mu.g/ml IGF-1 10 ng/ml EGF 10 ng/ml
NGF 200 ng/ml
TABLE 17 EuroCollins Solution NaHCO.sub.3 10 mM KCl 15 mM K.sub.2
HPO.sub.4 42.5 mM KH.sub.2 PO.sub.4 15.1 mM Glucose 195 mM
Substance P 10 .mu.g/ml IGF-1 10 ng/ml EGF 10 ng/ml
II. Uses of Media
It is contemplated that the media described above may be utilized
in a variety of transplant and other medical procedures. It is
contemplated that the media can be used for the preservation of any
tissue, organ, cell(s), or organisms, including, but not limited
to, organs, genetically engineered tissues, biomedically engineered
tissues, sperm, eggs, and embryos. In particular, the media finds
use for the preservation of both internal and external organs prior
to transplant. In some embodiments, the media is utilized for
hypothermic storage of the organ. In hypothermic storage, the organ
is flushed with the media, cooled, suspended in the media, and
stored. In other embodiments, the media is utilized for pulsatile
hypothermic perfusion of the organ. In still further embodiments,
the present invention provides a composition comprising an internal
organ suspended in or perfused with a media comprising one or more
antimicrobial polypeptides (e.g., defensins) and/or at least one
cell surface receptor binding protein. In particularly preferred
embodiments, the media of the present invention are useful for
decreasing the incidence and/or severity of delayed graft function
in patients receiving transplanted kidneys stored and/or treated
with the media of the present invention.
In other embodiments, the present invention provides a composition
comprising skin or another external organ suspended in or perfused
with a media comprising an antimicrobial peptide or other pore
forming agents and/or at least one growth factor. In other
embodiments, the media may be used in procedures such as
cardioplegia (See, e.g., U.S. Pat. No. 5,514,536, incorporated
herein by reference).
Experimental
The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
EXAMPLE 1
This Example describes the use of media comprising defensins for
the storage of organs prior to transplant. The study was performed
on adult beagle dogs of both sexes weighing approximately 8 kg. The
study employed a kidney autotransplantation with immediate
contralateral nephrectomy model. This involved harvesting of either
the left or right kidney and flushing it out through the renal
artery with the University of Wisconsin solution (See Table 3)
either with or without added defensins (1 mg/liter), storage of the
kidney under sterile conditions on ice for 3 days, reimplantation
of the previously harvested kidney into the abdominal cavity of the
same dog and then immediately removing the other kidney.
For harvest of the kidney, a midline abdominal incision was made
and the left kidney isolated by dissecting free of any attachments
to its artery, vein and ureter. The ureter was ligated with a
single 4-0 silk ligature near the bladder and divided proximal to
the ligature. The gonadal vein was ligated with 2 4-0 silk
ligatures and divided. The renal artery and vein were then clamped
and cut and the kidney removed for vascular flushing with
preservation solution and experimental storage. The kidney was then
suspended in preservation solution in sterile plastic bags and
placed on ice in a cooler for storage. The stumps of the renal
artery and vein were ligated separately with doubled 3-0 silk
ligatures. The excision site was inspected for hemorrhage and any
small bleeders were cauterized or ligated. The body wall was closed
with 0-Maxon in a simple continuous pattern. The skin was then
closed with 3-0 Vicryl in a simple continuous subcuticular pattern
after which the dog was recovered from anesthesia.
Three days after harvest of the kidney the dog was anesthetized for
reimplantation of the stored kidney. Intravenous morphine (0.5
mg/kg) was administered as prophylaxis against intussusception. The
abdomen was entered through a midline abdominal incision made by
opening the previous incision and extending the incision to the
pubis. The external iliac artery and common iliac vein were
isolated by blunt and sharp dissection. The external iliac artery
was ligated distally, clamped proximally with an atraumatic
vascular clamp and divided just proximal to the ligature. The free
arterial end was flushed with heparinized saline and its end
cleared of loose adventitia. The common iliac vein surface was
cleared of loose adventitia by sharp dissection. An atraumatic
vascular clamp was placed on the vein both proximally and distally
and the vein wall fenestrated using a Metzenbaum scissors. The vein
segment was flushed free of blood with heparinized saline. Four 7-0
polyester sutures were placed in the wall of the vein exiting the
fenestration and attached to the renal vein. The renal vein was
apposed to the side of the iliac vein and the anastomosis performed
using two lines (front and back vessel walls) of simple continuous
suture. The renal artery was apposed to the end of the external
iliac artery using two 7-0 polypropylene sutures and the
anastomosis completed with two lines of simple continuous suture.
The proximal venous clamp was removed followed by the arterial
vascular clamp. Mannitol (1 gm/kg IV) was administered during
anastomosis, which required less than 30 minutes to complete. The
bladder was entered through a ventral incision and fenestrated on
its dorsal side with a hemostat. The ureter was incised
longitudinally for 1 cm and then pulled through the bladder
fenestration. The ureteral mucosa and bladder mucosa was apposed
using 6-0 Vicryl suture in a continuous pattern. The bladder was
closed with 3-0 Vicryl in a Cushing pattern. The contralateral
kidney was excised and the ureter, renal artery, and renal vein
ligated with 3-0 silk. The abdominal wall was closed with 0-Maxon
and the skin with 3-0 Vicryl using continuous suture patterns in
the linea alba and subcuticular layers, respectively. The dog was
then given 500 ml lactated Ringer's solution subcutaneously and
recovered from anesthesia.
The results are presented in FIG. 1. As can be seen, dogs receiving
kidneys stored for three days in UW solution supplemented with
BNP-1 exhibited serum creatinine of about half that seen in dogs
receiving kidneys stored in UW solution alone. This is indicative
of markedly improved renal function in kidneys preserved in media
containing BNP-1.
EXAMPLE 2
This Example describes the use of media comprising defensins and/or
cell surface receptor binding compounds for the storage of organs
prior to transplant. The study was performed as described in
Example 1, except that the organs were stored for four days prior
to transplant. The three test groups were UW solution alone, UW
solution supplemented with 1 mg/L BNP-1 (synthesized by Multiple
Peptide Systems, San Diego Calif.), and UW solution supplemented
with 1 mg/L BNP-1, and the following trophic factors: 20 mM
trehalose (Sigma, St. Louis Mo.), 2.5 mg/L substance P (Sigma), 10
.mu.g/L IGF-1 (Collaborative Biologicals), 10 .mu.g/L EGF (Sigma),
and 200 ng/ml NGF (Sigma [murine] or Genentech [human])). The
results are presented in FIG. 2. As can be seen, dogs receiving
kidneys stored in UW solution supplemented with BNP-1 and cell
surface receptor binding compounds exhibited serum creatinine of
about half that seen in dogs receiving kidneys stored in UW
solution supplemented with BNP-1 or UW solution alone.
Surprisingly, the serum creatinine levels in the dogs receiving
kidneys stored in UW solution supplemented with both BNP-1 and cell
surface receptor binding compounds remarkably improved the quality
of preservation to the point that they equal 3 day BNP-1 preserved
kidneys and 2 day or less storage with UW solution alone.
EXAMPLE 3
This Example describes results from the transplant of kidneys after
six days of storage. This study was performed as described in
Example 1, except that the kidneys were stored for four days in UW
solution prior to transplant or six days in UW solution
supplemented with a defensin and trophic factors (See Example 2)
prior to transplant. The results are presented in FIG. 3. As can be
seen, the serum creatinine levels following transplant were similar
in the two groups. These data demonstrate that UW solution
supplemented with trophic factors can be used increase the duration
of storage.
EXAMPLE 4
This Example describes results from the transplant of kidneys after
six days of storage. This study was performed as described in
Example 3, except that the kidneys were stored for three days in UW
solution prior to transplant or six days in UW solution
supplemented with a defensin and trophic factors (See Example 2)
prior to transplant. The results are presented in FIG. 4. As can be
seen, the serum creatinine levels following transplant were higher
in the dogs receiving kidneys stored for six days as opposed dogs
receiving kidneys stored for three days. These data demonstrate
that UW solution supplemented with trophic factors can be used
increase the duration of storage.
EXAMPLE 5
This Example describes the results from the transplant of kidneys
after five days of storage. This study was performed as described
in Example 3, except that the kidneys were stored for three days in
UW solution prior to transplant or five days in UW solution
supplemented with a defensin and trophic factors (See Example 2)
prior to transplant. The results are presented in FIG. 5. As can be
seen, the serum creatinine levels following transplant were higher
in the dogs receiving kidneys stored for three days in UW solution
as opposed dogs receiving kidneys stored for five days in UW
solution plus trophic factors. These data demonstrate that UW
solution supplemented with trophic factors can be used increase the
duration of storage.
EXAMPLE 6
This Example describes the results from the transplant of kidneys
after four days of storage. This study was performed as described
in Example 3, except that the kidneys were stored for three days in
UW solution prior to transplant or four days in UW solution
supplemented with a defensin and trophic factors (See Example 2)
prior to transplant. The results are presented in FIG. 6. As can be
seen, the serum creatinine levels following transplant were
significantly higher in the dogs receiving kidneys stored for three
days in UW solution as opposed dogs receiving kidneys stored for
four days in UW solution plus trophic factors. These data
demonstrate that UW solution supplemented with trophic factors can
be used increase the duration of storage are indicative of markedly
improved renal function in kidneys preserved in media containing
trophic factors.
EXAMPLE 7
This Example describes the results from the transplant of kidneys
after four days of storage. This study was performed as described
in Example 3, except that the kidneys were stored for four days in
UW solution prior to transplant or four days in UW solution
supplemented with a defensin and trophic factors (See Example 2)
prior to transplant. The results are presented in FIG. 7. As can be
seen, the serum creatinine levels following transplant were
significantly higher in the dogs receiving kidneys stored for four
days in UW solution as opposed dogs receiving kidneys stored for
four days in UW solution plus trophic factors. These data are
indicative of markedly improved renal function in kidneys preserved
in media containing trophic factors.
EXAMPLE 8
This Example demonstrates that hydroxyethyl starch can be deleted
from UW solution without adversely affecting organ quality. This
study was performed as described in Example 3, except that the
kidneys were stored for five days prior to transplant in UW
solution containing hydroxyethyl starch and supplemented with
trophic factors or five days prior to transplant in UW solution
supplemented with trophic factors (See Example 2), and in which the
hydroxyethyl starch was omitted. The results are presented in FIG.
8. Surprisingly, the serum creatinine levels following transplant
were significantly higher in the dogs receiving kidneys stored in
UW solution containing hydroxyethyl starch as opposed dogs
receiving kidneys stored in UW solution without hydroxyethyl
starch.
EXAMPLE 9
This Example demonstrates experiments where use of the D-form
isomer of BNP-1 was compared with L-form isomer. The D-form isomers
was synthesized with D-amino acids. This study was performed as
described in Example 1, except that the kidneys were stored for
three days prior to transplant in UW solution containing the L-form
isomer of BNP-1 or three days prior to transplant in UW solution
containing the D-form isomer of BNP-1. The results are presented in
FIG. 9. As can be seen, dogs receiving kidneys stored in media
supplemented with the D-form isomer returned to normal serum
creatinine levels faster than dogs receiving kidneys stored in the
media supplemented with the L-form isomer.
EXAMPLE 10
This Example describes the effect UW solution supplemented with
BNP-1 on cytoskeletal structure of kidney cells. Briefly, either
MDCK cells or primary kidney cell cultures were stored for three
days at cold temperatures in either UW solution, UW solution
supplemented with BNP-1, or DMEM. The cells were then labeled with
actin and tubulin antibodies and analyzed by confocal fluorescence
microscopy. Control untreated cells displayed a homogeneous fine
fibrillar pattern of actin and tubulin that extended throughout the
cell. Cells stored in DMEM culture media at cold temperatures
displayed nearly complete dissolution of both actin and tubulin
with very little staining present. Cells stored in UW solution had
nearly complete disruption of the tubulin elements and significant
dissolution of the actin microfilaments. In primary cultures in UW
solution, the residual actin in condensed along the plasma
membrane. Treatment with BNP-1 during storage resulted in better
maintenance of actin and tubulin in MDCK cells. In primary cultures
with BNP-1, the tubulin and actin were better stained and more
persistent with some condensation along stellate rays which
extended from the nucleus out to the plasma membrane of the
cells.
In a separate experiment, the effect of BNP-1 on the cytoskeleton
after three days cold storage in UW solution followed by 3 hours
warm reperfusion in DMEM culture media with 10% serum was
determined. MDCK cells stored in DMEM culture media at 4.degree. C.
failed to reassemble the cytoskeleton by 3 hours of reperfusion.
MDCK cells stored in UW solution and then reperfused were able to
reassemble the cytoskeleton, but in primary kidney cell cultures
the cytoskeleton remained abnormal at 3 hours of reperfusion. In
these primary cells, the actin and tubulin filaments maintained a
coarse clumpy pattern with considerable cortical condensation near
the plasma membrane and only a limited amount of fine fibrillar
structure that would be considered more normal. Cells stored in
BNP-1 supplemented UW solution and reperfused had superior
maintenance and reassembly of the cytoskeleton in both MDCK and
primary renal cultures with homogeneously distributed fine
fibrillar cytoskeletal elements predominating in these cells.
All publications and patents mentioned in the above specification
are herein incorporated by reference. Various modifications and
variations of the described method and system of the invention will
be apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in organ storgae and transplant,
cryobiology, biochemistry, or related fields are intended to be
within the scope of the following claims.
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<210> SEQ ID NO 1 <211> LENGTH: 64 <212> TYPE:
PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 1 Met
Arg Leu His His Leu Leu Leu Ala Leu Leu Phe Leu Val Leu Ser 1 5 10
15 Ala Gly Ser Gly Phe Thr Gln Gly Val Arg Asn Ser Gln Ser Cys Arg
20 25 30 Arg Asn Lys Gly Ile Cys Val Pro Ile Arg Cys Pro Gly Ser
Met Arg 35 40 45 Gln Ile Gly Thr Cys Leu Gly Ala Gln Val Lys Cys
Cys Arg Arg Lys 50 55 60 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2 <211> LENGTH: 24 <212> TYPE:
PRT <213> ORGANISM: Xenopus laevis <400> SEQUENCE: 2
Gly Val Leu Ser Asn Val Ile Gly Tyr Leu Lys Lys Leu Gly Thr Gly 1 5
10 15 Ala Leu Asn Ala Val Leu Lys Gln 20 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 81
<212> TYPE: PRT <213> ORGANISM: Xenopus laevis
<400> SEQUENCE: 3 Met Tyr Lys Gly Ile Phe Leu Cys Val Leu Leu
Ala Val Ile Cys Ala 1 5 10 15 Asn Ser Leu Ala Thr Pro Ser Ser Asp
Ala Asp Glu Asp Asn Asp Glu 20 25 30 Val Glu Arg Tyr Val Arg Gly
Trp Ala Ser Lys Ile Gly Gln Thr Leu 35 40 45 Gly Lys Ile Ala Lys
Val Gly Leu Lys Glu Leu Ile Gln Pro Lys Arg 50 55 60 Glu Ala Met
Leu Arg Ser Ala Glu Ala Gln Gly Lys Arg Pro Trp Ile 65 70 75 80 Leu
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4
<211> LENGTH: 303 <212> TYPE: PRT <213> ORGANISM:
Xenopus laevis <400> SEQUENCE: 4 Met Phe Lys Gly Leu Phe Ile
Cys Ser Leu Ile Ala Val Ile Cys Ala 1 5 10 15 Asn Ala Leu Pro Gln
Pro Glu Ala Ser Ala Asp Glu Asp Met Asp Glu 20 25 30 Arg Glu Val
Arg Gly Ile Gly Lys Phe Leu His Ser Ala Gly Lys Phe 35 40 45 Gly
Lys Ala Phe Val Gly Glu Ile Met Lys Ser Lys Arg Asp Ala Glu 50 55
60 Ala Val Gly Pro Glu Ala Phe Ala Asp Glu Asp Leu Asp Glu Arg Glu
65 70 75 80 Val Arg Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe
Gly Lys 85 90 95 Ala Phe Val Gly Glu Ile Met Asn Ser Lys Arg Asp
Ala Glu Ala Val 100 105 110 Gly Pro Glu Ala Phe Ala Asp Glu Asp Leu
Asp Glu Arg Glu Val Arg 115 120 125 Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Phe 130 135 140 Val Gly Glu Ile Met Asn
Ser Lys Arg Asp Ala Glu Ala Val Gly Pro 145 150 155 160 Glu Ala Phe
Ala Asp Glu Asp Leu Asp Glu Arg Glu Val Arg Gly Ile 165 170 175 Gly
Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe Val Gly 180 185
190 Glu Ile Met Asn Ser Lys Arg Asp Ala Glu Ala Val Gly Pro Glu Ala
195 200 205 Phe Ala Asp Glu Asp Phe Asp Glu Arg Glu Val Arg Gly Ile
Gly Lys 210 215 220 Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe
Val Gly Glu Ile 225 230 235 240 Met Asn Ser Lys Arg Asp Ala Glu Ala
Val Gly Pro Glu Ala Phe Ala 245 250 255 Asp Glu Asp Leu Asp Glu Arg
Glu Val Arg Gly Ile Gly Lys Phe Leu 260 265 270 His Ser Ala Lys Lys
Phe Gly Lys Ala Phe Val Gly Glu Ile Met Asn 275 280 285 Ser Lys Arg
Asp Ala Glu Ala Val Asp Asp Arg Arg Trp Val Glu 290 295 300
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Tachypleus gigas <400> SEQUENCE: 5 Lys Trp Cys Phe Arg Val
Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys 1 5 10 15 Arg <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Tachypleus
gigas <400> SEQUENCE: 6 Arg Trp Cys Phe Arg Val Cys Tyr Arg
Gly Ile Cys Tyr Arg Lys Cys 1 5 10 15 Arg <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 129
<212> TYPE: PRT <213> ORGANISM: Bufo gargarizans
<400> SEQUENCE: 7 Met Ser Gly Arg Gly Lys Gln Gly Gly Lys Val
Arg Ala Lys Ala Lys 1 5 10 15 Thr Arg Ser Ser Arg Ala Gly Leu Gln
Phe Pro Val Gly Arg Val His 20 25 30 Arg Leu Leu Arg Lys Gly Asn
Tyr Ala Gln Arg Val Gly Ala Gly Ala 35 40 45 Pro Val Tyr Leu Ala
Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu 50 55 60 Glu Leu Ala
Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile 65 70 75 80 Pro
Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys 85 90
95 Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110 Gln Ala Val Leu Leu Pro Lys Thr Glu Ser Ser Lys Pro Ala
Lys Ser 115 120 125 Lys <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Bufo gargarizans <400> SEQUENCE: 8
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His 1 5
10 15 Arg Leu Leu Arg Lys 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9 <211> LENGTH: 63 <212> TYPE:
PRT <213> ORGANISM: Bombyx mori <400> SEQUENCE: 9 Met
Asn Phe Val Arg Ile Leu Ser Phe Val Phe Ala Leu Val Leu Ala 1 5 10
15 Leu Gly Ala Val Ser Ala Ala Pro Glu Pro Arg Trp Lys Leu Phe Lys
20 25 30 Lys Ile Glu Lys Val Gly Arg Asn Val Arg Asp Gly Leu Ile
Lys Ala 35 40 45 Gly Pro Ala Ile Ala Val Ile Gly Gln Ala Lys Ser
Leu Gly Lys 50 55 60 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 10 <211> LENGTH: 63 <212> TYPE:
PRT <213> ORGANISM: Bombyx mori <400> SEQUENCE: 10 Met
Asn Phe Ala Lys Ile Leu Ser Phe Val Phe Ala Leu Val Leu Ala 1 5 10
15 Leu Ser Met Thr Ser Ala Ala Pro Glu Pro Arg Trp Lys Ile Phe Lys
20 25 30 Lys Ile Glu Lys Met Gly Arg Asn Ile Arg Asp Gly Ile Val
Lys Ala 35 40 45 Gly Pro Ala Ile Glu Val Leu Gly Ser Ala Lys Ala
Ile Gly Lys 50 55 60 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11 <211> LENGTH: 63 <212> TYPE:
PRT <213> ORGANISM: Drosophila melanogaster <400>
SEQUENCE: 11 Met Asn Phe Tyr Lys Ile Phe Val Phe Val Ala Leu Ile
Leu Ala Ile 1 5 10 15 Ser Ile Gly Gln Ser Glu Ala Gly Trp Leu Lys
Lys Leu Gly Lys Arg 20 25 30 Ile Glu Arg Ile Gly Gln His Thr Arg
Asp Ala Thr Ile Gln Gly Leu 35 40 45 Gly Ile Ala Gln Gln Ala Ala
Asn Val Ala Ala Thr Ala Arg Gly 50 55 60 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Sus scrofa <400>
SEQUENCE: 12 Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser
Ala Lys Lys 1 5 10 15 Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln
Gly Gly Pro Arg 20 25 30 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 13 Ile
Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14 <211>
LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Lactococcus
lactis <400> SEQUENCE: 14 Ile Thr Ser Ile Ser Leu Cys Thr Pro
Gly Cys Lys Thr Gly Ala Leu 1 5 10 15 Met Gly Cys Asn Met Lys Thr
Ala Thr Cys His Cys Ser Ile His Val 20 25 30 Ser Lys <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Rana
catesbeiana <400> SEQUENCE: 15 Phe Leu Gly Gly Leu Ile Lys
Ile Val Pro Ala Met Ile Cys Ala Val 1 5 10 15 Thr Lys Lys Cys 20
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Bos taurus <400> SEQUENCE: 16 Phe Lys Cys Arg Arg Trp Gln Trp
Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val Arg
Arg Ala Phe 20 25 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 17 <211> LENGTH: 19 <212> TYPE: PRT
<213> ORGANISM: Sus scrofa <220> FEATURE: <221>
NAME/KEY: SITE <222> LOCATION: (19) <223> OTHER
INFORMATION: Xaa at this position can be any amino acid.
<400> SEQUENCE: 17 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg
Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg Xaa <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 18 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Sus scrofa <400>
SEQUENCE: 18 Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Ile
Cys Val Gly 1 5 10 15
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19
<211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 19 Met Lys Phe Phe Val Phe Ala
Leu Ile Leu Ala Leu Met Leu Ser Met 1 5 10 15 Thr Gly Ala Asp Ser
His Ala Lys Arg His His Gly Tyr Lys Arg Lys 20 25 30 Phe His Glu
Lys His His Ser His Arg Gly Tyr Arg Ser Asn Tyr Leu 35 40 45 Tyr
Asp Asn 50 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 20 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Macaca fascicularis <400> SEQUENCE: 20 Asp Ser His
Glu Glu Arg His His Gly Arg His Gly His His Lys Tyr 1 5 10 15 Gly
Arg Lys Phe His Glu Lys His His Ser His Arg Gly Tyr Arg Ser 20 25
30 Asn Tyr Leu Tyr Asp Asn 35 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 21 <211> LENGTH: 33 <212> TYPE:
PRT <213> ORGANISM: Phyllomedusa sauvagei <400>
SEQUENCE: 21 Ala Leu Trp Lys Thr Met Leu Lys Lys Leu Gly Thr Met
Ala Leu His 1 5 10 15 Ala Gly Lys Ala Ala Leu Gly Ala Ala Ala Asp
Thr Ile Ser Gln Thr 20 25 30 Gln <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 22 <211> LENGTH: 34
<212> TYPE: PRT <213> ORGANISM: Phyllomedusa sauvagei
<400> SEQUENCE: 22 Ala Leu Trp Phe Thr Met Leu Lys Lys Leu
Gly Thr Met Ala Leu His 1 5 10 15 Ala Gly Lys Ala Ala Leu Gly Ala
Ala Ala Asn Thr Ile Ser Gln Gly 20 25 30 Thr Gln <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 23 <211>
LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Phyllomedusa
sauvagei <400> SEQUENCE: 23 Ala Leu Trp Lys Asn Met Leu Lys
Gly Ile Gly Lys Leu Ala Gly Lys 1 5 10 15 Ala Ala Leu Gly Ala Val
Lys Lys Leu Val Gly Ala Glu Ser 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 24 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Misgurnus
Anguillicaudatus <400> SEQUENCE: 24 Arg Gln Arg Val Glu Glu
Leu Ser Lys Phe Ser Lys Lys Gly Ala Ala 1 5 10 15 Ala Arg Arg Arg
Lys 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
25 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Apis mellifera <400> SEQUENCE: 25 Gly Ile Gly Ala
Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser
Trp Ile Ser Arg Lys Lys Arg Gln Gln 20 25 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 26 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Pardachirus pavoninus
<400> SEQUENCE: 26 Gly Phe Phe Ala Leu Ile Pro Lys Ile Ile
Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser
Ala Leu Ser Ser Ser Gly Glu Gln 20 25 30 Glu <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 27 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Pardachirus pavoninus
<400> SEQUENCE: 27 Gly Phe Phe Ala Leu Ile Pro Lys Ile Ile
Ser Ser Pro Ile Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser
Ala Leu Ser Ser Ser Gly Gly Gln 20 25 30 Glu <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 28 <211> LENGTH: 176
<212> TYPE: PRT <213> ORGANISM: Bos taurus <400>
SEQUENCE: 28 Met Glu Thr Gln Arg Ala Ser Leu Ser Leu Gly Arg Cys
Ser Leu Trp 1 5 10 15 Leu Leu Leu Leu Gly Leu Val Leu Pro Ser Ala
Ser Ala Gln Ala Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala
Val Asp Gln Phe Asn Glu Arg 35 40 45 Ser Ser Glu Ala Asn Leu Tyr
Arg Leu Leu Glu Leu Asp Pro Thr Pro 50 55 60 Asn Asp Asp Leu Asp
Pro Gly Thr Arg Lys Pro Val Ser Phe Arg Val 65 70 75 80 Lys Glu Thr
Asp Cys Pro Arg Thr Ser Gln Gln Pro Leu Glu Gln Cys 85 90 95 Asp
Phe Lys Glu Asn Gly Leu Val Lys Gln Cys Val Gly Thr Val Thr 100 105
110 Leu Asp Pro Ser Asn Asp Gln Phe Asp Ile Asn Cys Asn Glu Leu Gln
115 120 125 Ser Val Arg Phe Arg Pro Pro Ile Arg Arg Pro Pro Ile Arg
Pro Pro 130 135 140 Phe Tyr Pro Pro Phe Arg Pro Pro Ile Arg Pro Pro
Ile Phe Pro Pro 145 150 155 160 Ile Arg Pro Pro Phe Arg Pro Pro Leu
Gly Pro Phe Pro Gly Arg Arg 165 170 175 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 29 <211> LENGTH: 155
<212> TYPE: PRT <213> ORGANISM: Bos taurus <400>
SEQUENCE: 29 Met Glu Thr Pro Arg Ala Ser Leu Ser Leu Gly Arg Trp
Ser Leu Trp 1 5 10 15 Leu Leu Leu Leu Gly Leu Ala Leu Pro Ser Ala
Ser Ala Gln Ala Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala
Val Asp Gln Leu Asn Glu Gln 35 40 45 Ser Ser Glu Pro Asn Ile Tyr
Arg Leu Leu Glu Leu Asp Gln Pro Pro 50 55 60 Gln Asp Asp Glu Asp
Pro Asp Ser Pro Lys Arg Val Ser Phe Arg Val 65 70 75 80 Lys Glu Thr
Val Cys Ser Arg Thr Thr Gln Gln Pro Pro Glu Gln Cys 85 90 95 Asp
Phe Lys Glu Asn Gly Leu Leu Lys Arg Cys Glu Gly Thr Val Thr 100 105
110 Leu Asp Gln Val Arg Gly Asn Phe Asp Ile Thr Cys Asn Asn His Gln
115 120 125 Ser Ile Arg Ile Thr Lys Gln Pro Trp Ala Pro Pro Gln Ala
Ala Arg 130 135 140 Leu Cys Arg Ile Val Val Ile Arg Val Cys Arg 145
150 155 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
30 <211> LENGTH: 29 <212> TYPE: PRT <213>
ORGANISM: Ceratitis capitata <400> SEQUENCE: 30 Ser Ile Gly
Ser Ala Leu Lys Lys Ala Leu Pro Val Ala Lys Lys Ile 1 5 10 15 Gly
Lys Ile Ala Leu Pro Ile Ala Lys Ala Ala Leu Pro 20 25 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 31 <211>
LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Ceratitis
capitata <400> SEQUENCE: 31 Ser Ile Gly Ser Ala Phe Lys Lys
Ala Leu Pro Val Ala Lys Lys Ile 1 5 10 15 Gly Lys Ala Ala Leu Pro
Ile Ala Lys Ala Ala Leu Pro 20 25 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 32 <211> LENGTH: 170
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 32 Met Lys Thr Gln Arg Asn Gly His Ser Leu
Gly Arg Trp Ser Leu Val 1 5 10 15 Leu Leu Leu Leu Gly Leu Val Met
Pro Leu Ala Ile Ile Ala Gln Val 20 25 30 Leu Ser Tyr Lys Glu Ala
Val Leu Arg Ala Ile Asp Gly Ile Asn Gln 35 40 45 Arg Ser Ser Asp
Ala Asn Leu Tyr Arg Leu Leu Asp Leu Asp Pro Arg 50 55 60 Pro Thr
Met Asp Gly Asp Pro Asp Thr Pro Lys Pro Val Ser Phe Thr 65 70 75 80
Val Lys Glu Thr Val Cys Pro Arg Thr Thr Gln Gln Ser Pro Glu Asp 85
90 95 Cys Asp Phe Lys Lys Asp Gly Leu Val Lys Arg Cys Met Gly Thr
Val 100 105 110 Thr Leu Asn Gln Ala Arg Gly Ser Phe Asp Ile Ser Cys
Asp Lys Asp 115 120 125 Asn Lys Arg Phe Ala Leu Leu Gly Asp Phe Phe
Arg Lys Ser Lys Glu 130 135 140 Lys Ile Gly Lys Glu Phe Lys Arg Ile
Val Gln Arg Ile Lys Asp Phe 145 150 155 160 Leu Arg Asn Leu Val Pro
Arg Thr Glu Ser 165 170 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 33 <211> LENGTH: 170 <212> TYPE:
PRT <213> ORGANISM: Equus caballus <400> SEQUENCE: 33
Met Glu Thr Gln Arg Asn Thr Arg Cys Leu Gly Arg Trp Ser Pro Leu 1 5
10 15 Leu Leu Leu Leu Gly Leu Val Ile Pro Pro Ala Thr Thr Gln Ala
Leu 20 25 30 Ser Tyr Lys Glu Ala Val Leu Arg Ala Val Asp Gly Leu
Asn Gln Arg 35 40 45 Ser Ser Asp Glu Asn Leu Tyr Arg Leu Leu Glu
Leu Asp Pro Leu Pro 50 55 60 Lys Gly Asp Lys Asp Ser Asp Thr Pro
Lys Pro Val Ser Phe Met Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg
Ile Met Lys Gln Thr Pro Glu Gln Cys 85 90 95 Asp Phe Lys Glu Asn
Gly Leu Val Lys Gln Cys Val Gly Thr Val Ile 100 105 110 Leu Asp Pro
Val Lys Asp Tyr Phe Asp Ala Ser Cys Asp Glu Pro Gln 115 120 125 Arg
Val Lys Arg Phe His Ser Val Gly Ser Leu Ile Gln Arg His Gln 130 135
140 Gln Met Ile Arg Asp Lys Ser Glu Ala Thr Arg His Gly Ile Arg Ile
145 150 155 160 Ile Thr Arg Pro Lys Leu Leu Leu Ala Ser 165 170
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 34
<211> LENGTH: 159 <212> TYPE: PRT <213> ORGANISM:
Bos taurus <400> SEQUENCE: 34 Met Glu Thr Gln Arg Ala Ser Leu
Ser Leu Gly Arg Trp Ser Leu Trp 1 5 10 15 Leu Leu Leu Leu Gly Leu
Ala Leu Pro Ser Ala Ser Ala Gln Ala Leu 20 25 30 Ser Tyr Arg Glu
Ala Val Leu Arg Ala Val Asp Gln Leu Asn Glu Lys 35 40 45 Ser Ser
Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp Pro Pro Pro 50 55 60
Lys Glu Asp Asp Glu Asn Pro Asn Ile Pro Lys Pro Val Ser Phe Arg 65
70 75 80 Val Lys Glu Thr Val Cys Pro Arg Thr Ser Gln Gln Ser Pro
Glu Gln 85 90 95 Cys Asp Phe Lys Glu Asn Gly Leu Leu Lys Glu Cys
Val Gly Thr Val 100 105 110 Thr Leu Asp Gln Val Gly Ser Asn Phe Asp
Ile Thr Cys Ala Val Pro
115 120 125 Gln Ser Val Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu
Arg Ala 130 135 140 Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile
Arg Ile Gly 145 150 155 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 35 <211> LENGTH: 156 <212> TYPE:
PRT <213> ORGANISM: Equus asinus <400> SEQUENCE: 35 Met
Glu Thr Gln Arg Asn Thr Arg Cys Leu Gly Arg Trp Ser Pro Leu 1 5 10
15 Leu Leu Leu Leu Gly Leu Val Ile Pro Pro Ala Thr Thr Gln Ala Leu
20 25 30 Ser Tyr Lys Glu Ala Val Leu Arg Ala Val Asp Gly Leu Asn
Gln Arg 35 40 45 Ser Ser Asp Glu Asn Leu Tyr Arg Leu Leu Glu Leu
Asp Pro Leu Pro 50 55 60 Lys Gly Asp Lys Asp Ser Asp Thr Pro Lys
Pro Val Ser Phe Met Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg Ile
Met Lys Gln Thr Pro Glu Gln Cys 85 90 95 Asp Phe Lys Glu Asn Gly
Leu Val Lys Gln Cys Val Gly Thr Val Ile 100 105 110 Leu Gly Pro Val
Lys Asp His Phe Asp Val Ser Cys Gly Glu Pro Gln 115 120 125 Arg Val
Lys Arg Phe Gly Arg Leu Ala Lys Ser Phe Leu Arg Met Arg 130 135 140
Ile Leu Leu Pro Arg Arg Lys Ile Leu Leu Ala Ser 145 150 155
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 36
<211> LENGTH: 160 <212> TYPE: PRT <213> ORGANISM:
Ovis aries <400> SEQUENCE: 36 Met Glu Thr Gln Arg Ala Ser Leu
Ser Leu Gly Arg Cys Ser Leu Trp 1 5 10 15 Leu Leu Leu Leu Gly Leu
Ala Leu Pro Ser Ala Ser Ala Gln Val Leu 20 25 30 Ser Tyr Arg Glu
Ala Val Leu Arg Ala Ala Asp Gln Leu Asn Glu Lys 35 40 45 Ser Ser
Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp Pro Pro Pro 50 55 60
Lys Gln Asp Asp Glu Asn Ser Asn Ile Pro Lys Pro Val Ser Phe Arg 65
70 75 80 Val Lys Glu Thr Val Cys Pro Arg Thr Ser Gln Gln Pro Ala
Glu Gln 85 90 95 Cys Asp Phe Lys Glu Asn Gly Leu Leu Lys Glu Cys
Val Gly Thr Val 100 105 110 Thr Leu Asp Gln Val Arg Asn Asn Phe Asp
Ile Thr Cys Ala Glu Pro 115 120 125 Gln Ser Val Arg Gly Leu Arg Arg
Leu Gly Arg Lys Ile Ala His Gly 130 135 140 Val Lys Lys Tyr Gly Pro
Thr Val Leu Arg Ile Ile Arg Ile Ala Gly 145 150 155 160 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 37 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Bos taurus
<400> SEQUENCE: 37 Arg Leu Cys Arg Ile Val Val Ile Arg Val
Cys Arg 1 5 10 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 38 <211> LENGTH: 30 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 38 Ala Cys
Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15
Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 39
<211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 39 Cys Tyr Cys Arg Ile Pro Ala
Cys Ile Ala Gly Glu Arg Arg Tyr Gly 1 5 10 15 Thr Cys Ile Tyr Gln
Gly Arg Leu Trp Ala Phe Cys Cys 20 25 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 40 <211> LENGTH: 30
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 40 Asp Cys Tyr Cys Arg Ile Pro Ala Cys Ile
Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr Gln Gly Arg
Leu Trp Ala Phe Cys Cys 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 41 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 41 Val Cys Ser Cys Arg Leu Val Phe Cys Arg
Arg Thr Glu Leu Arg Val 1 5 10 15 Gly Asn Cys Leu Ile Gly Gly Val
Ser Phe Thr Tyr Cys Cys Thr Arg 20 25 30 Val <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 42 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 42 Val Val Cys Ala Cys Arg Arg Ala Leu Cys
Leu Pro Arg Glu Arg Arg 1 5 10 15 Ala Gly Phe Cys Arg Ile Arg Gly
Arg Ile His Pro Leu Cys Cys Arg 20 25 30 Arg <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 43 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 43 Val Val Cys Ala Cys Arg Arg Ala Leu Cys
Leu Pro Leu Glu Arg Arg 1 5 10 15 Ala Gly Phe Cys Arg Ile Arg Gly
Arg Ile His Pro Leu Cys Cys Arg 20 25 30 Arg <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 44 <211> LENGTH: 34
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 44 Gly Ile Cys Ala Cys Arg Arg Arg Phe Cys
Pro Asn Ser Glu Arg Phe 1 5 10 15 Ser Gly Tyr Cys Arg Val Asn Gly
Ala Arg Tyr Val Arg Cys Cys Ser 20 25 30 Arg Arg <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 45 <211>
LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Oryctolagus
cuniculus <400> SEQUENCE: 45 Gly Arg Cys Val Cys Arg Lys Gln
Leu Leu Cys Ser Tyr Arg Glu Arg 1 5 10 15 Arg Ile Gly Asp Cys Lys
Ile Arg Gly Val Arg Phe Pro Phe Cys Cys 20 25 30 Pro Arg
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 46
<211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM:
Oryctolagus cuniculus <400> SEQUENCE: 46 Val Ser Cys Thr Cys
Arg Arg Phe Ser Cys Gly Phe Gly Glu Arg Ala 1 5 10 15 Ser Gly Ser
Cys Thr Val Asn Gly Gly Val Arg His Thr Leu Cys Cys 20 25 30 Arg
Arg <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 47
<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM:
Oryctolagus cuniculus <400> SEQUENCE: 47 Val Phe Cys Thr Cys
Arg Gly Phe Leu Cys Gly Ser Gly Glu Arg Ala 1 5 10 15 Ser Gly Ser
Cys Thr Ile Asn Gly Val Arg His Thr Leu Cys Cys Arg 20 25 30 Arg
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 48
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Rattus norvegicus <400> SEQUENCE: 48 Val Thr Cys Tyr Cys Arg
Arg Thr Arg Cys Gly Phe Arg Glu Arg Leu 1 5 10 15 Ser Gly Ala Cys
Gly Tyr Arg Gly Arg Ile Tyr Arg Leu Cys Cys Arg 20 25 30
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 49
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Rattus norvegicus <400> SEQUENCE: 49 Cys Ser Cys Arg Tyr Ser
Ser Cys Arg Phe Gly Glu Arg Leu Leu Ser 1 5 10 15 Gly Ala Cys Arg
Leu Asn Gly Arg Ile Tyr Arg Leu Cys Cys 20 25 30 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 50 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Rattus
norvegicus <400> SEQUENCE: 50 Ala Cys Thr Cys Arg Ile Gly Ala
Cys Val Ser Gly Glu Arg Leu Thr 1 5 10 15 Gly Ala Cys Gly Leu Asn
Gly Arg Ile Tyr Arg Leu Cys Cys Arg 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 51 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Guinea pig
cytomegalovirus <400> SEQUENCE: 51 Arg Arg Cys Ile Cys Thr
Thr Arg Thr Cys Arg Phe Pro Tyr Arg Arg 1 5 10 15 Leu Gly Thr Cys
Ile Phe Gln Asn Arg Val Tyr Thr Phe Cys Cys 20 25 30 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 52 <211>
LENGTH: 67 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 52 Met Arg Ile His Tyr Leu Leu Phe Ala Leu
Leu Phe Leu Phe Leu Val 1 5 10 15 Pro Val Pro Gly His Gly Gly Ile
Ile Asn Thr Leu Gln Lys Tyr Tyr 20 25 30 Cys Arg Val Arg Gly Gly
Arg Cys Ala Val Leu Ser Cys Leu Pro Lys 35 40 45 Glu Glu Gln Ile
Gly Lys Cys Ser Thr Arg Gly Arg Lys Cys Cys Arg 50 55 60 Arg Lys
Lys 65 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
53 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Macaca mulatta <400> SEQUENCE: 53 Arg Cys Ile Cys
Thr Arg Gly Phe Cys Arg Cys Leu Cys Arg Arg Gly 1 5 10 15 Val Cys
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 54
<211> LENGTH: 78 <212> TYPE: PRT <213> ORGANISM:
Helianthus annuus <400> SEQUENCE: 54 Met Lys Ser Ser Met Lys
Met Phe Ala Ala Leu Leu Leu Val Val Met 1 5 10 15 Cys Leu Leu Ala
Asn Glu Met Gly Gly Pro Leu Val Val Glu Ala Arg 20 25 30 Thr Cys
Glu Ser Gln Ser His Lys Phe Lys Gly Thr Cys Leu Ser Asp 35 40 45
Thr Asn Cys Ala Asn Val Cys His Ser Glu Arg Phe Ser Gly Gly Lys 50
55 60
Cys Arg Gly Phe Arg Arg Arg Cys Phe Cys Thr Thr His Cys 65 70 75
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 55
<211> LENGTH: 78 <212> TYPE: PRT <213> ORGANISM:
Helianthus annuus <400> SEQUENCE: 55 Met Lys Ser Ser Met Lys
Met Phe Ala Ala Leu Leu Leu Val Val Met 1 5 10 15 Cys Leu Leu Ala
Asn Glu Met Gly Gly Pro Leu Val Val Glu Ala Arg 20 25 30 Thr Cys
Glu Ser Gln Ser His Lys Phe Lys Gly Thr Cys Leu Ser Asp 35 40 45
Thr Asn Cys Ala Asn Val Cys His Ser Glu Arg Phe Ser Gly Gly Lys 50
55 60 Cys Arg Gly Phe Arg Arg Arg Cys Phe Cys Thr Thr His Cys 65 70
75 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 56
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Macaca mulatta <400> SEQUENCE: 56 Ala Cys Tyr Cys Arg Ile Pro
Ala Cys Leu Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Phe Tyr
Met Gly Arg Val Trp Ala Phe Cys Cys 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 57 <211> LENGTH: 37
<212> TYPE: PRT <213> ORGANISM: Androctonus Australis
Hector <400> SEQUENCE: 57 Gly Phe Gly Cys Pro Phe Asn Gln Gly
Ala Cys His Arg His Cys Arg 1 5 10 15 Ser Ile Arg Arg Arg Gly Gly
Tyr Cys Ala Gly Leu Phe Lys Gln Thr 20 25 30 Cys Thr Cys Tyr Arg 35
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 58
<211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM:
Mytilus galloprovincialis <220> FEATURE: <221>
NAME/KEY: SITE <222> LOCATION: (28) <223> OTHER
INFORMATION: Xaa at this position can be any amino acid.
<400> SEQUENCE: 58 Gly Phe Gly Cys Pro Asn Asn Tyr Gln Cys
His Arg His Cys Lys Ser 1 5 10 15 Ile Pro Gly Arg Cys Gly Gly Tyr
Cys Gly Gly Xaa His Arg Leu Arg 20 25 30 Cys Thr Cys Tyr Arg Cys 35
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 59
<211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM:
Heuchera sanguinea <400> SEQUENCE: 59 Asp Gly Val Lys Leu Cys
Asp Val Pro Ser Gly Thr Trp Ser Gly His 1 5 10 15 Cys Gly Ser Ser
Ser Lys Cys Ser Gln Gln Cys Lys Asp Arg Glu His 20 25 30 Phe Ala
Tyr Gly Gly Ala Cys His Tyr Gln Phe Pro Ser Val Lys Cys 35 40 45
Phe Cys Lys Arg Gln Cys 50 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 60 <211> LENGTH: 49 <212> TYPE:
PRT <213> ORGANISM: Clitoria ternatea <400> SEQUENCE:
60 Asn Leu Cys Glu Arg Ala Ser Leu Thr Trp Thr Gly Asn Cys Gly Asn
1 5 10 15 Thr Gly His Cys Asp Thr Gln Cys Arg Asn Trp Glu Ser Ala
Lys His 20 25 30 Gly Ala Cys His Lys Arg Gly Asn Trp Lys Cys Phe
Cys Tyr Phe Asn 35 40 45 Cys <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 61 <211> LENGTH: 91 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 61 Met
Lys Lys Leu Val Leu Leu Phe Ala Leu Val Leu Leu Ala Phe Gln 1 5 10
15 Val Gln Ala Asp Ser Ile Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu
20 25 30 Glu Gln Pro Gly Glu Lys Asp Gln Ala Val Ser Val Ser Phe
Gly Asp 35 40 45 Pro Gln Gly Ser Ala Leu Gln Asp Ala Ala Leu Gly
Trp Gly Arg Arg 50 55 60 Cys Pro Gln Cys Pro Arg Cys Pro Ser Cys
Pro Ser Cys Pro Arg Cys 65 70 75 80 Pro Arg Cys Pro Arg Cys Lys Cys
Asn Pro Lys 85 90 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 62 <211> LENGTH: 40 <212> TYPE: PRT
<213> ORGANISM: Bos taurus <400> SEQUENCE: 62 Gln Gly
Val Arg Asn Phe Val Thr Cys Arg Ile Asn Arg Gly Phe Cys 1 5 10 15
Val Pro Ile Arg Cys Pro Gly His Arg Arg Gln Ile Gly Thr Cys Leu 20
25 30 Gly Pro Gln Ile Lys Cys Cys Arg 35 40 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 63 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Bos taurus <400>
SEQUENCE: 63 Gln Gly Val Arg Asn Phe Val Thr Cys Arg Ile Asn Arg
Gly Phe Cys 1 5 10 15 Val Pro Ile Arg Cys Pro Gly His Arg Arg Gln
Ile Gly Thr Cys Leu 20 25 30 Gly Pro Arg Ile Lys Cys Cys Arg 35 40
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 64
<211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:
Bos taurus <400> SEQUENCE: 64 Gln Gly Val Arg Asn His Val Thr
Cys Arg Ile Tyr Gly Gly Phe Cys 1 5 10 15 Val Pro Ile Arg Cys Pro
Gly Arg Thr Arg Gln Ile Gly Thr Cys Phe 20 25 30 Gly Arg Pro Val
Lys Cys Cys Arg Arg Trp 35 40 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 65 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 65 Gln
Val Val Arg Asn Pro Gln Ser Cys Arg Trp Asn Met Gly Val Cys 1 5 10
15 Ile Pro Ile Ser Cys Pro Gly Asn Met Arg Gln Ile Gly Thr Cys Phe
20 25 30 Gly Pro Arg Val Pro Cys Cys Arg 35 40 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 66 <211> LENGTH: 41
<212> TYPE: PRT <213> ORGANISM: Bos taurus <400>
SEQUENCE: 66 Gln Arg Val Arg Asn Pro Gln Ser Cys Arg Trp Asn Met
Gly Val Cys 1 5 10 15 Ile Pro Phe Leu Cys Arg Val Gly Met Arg Gln
Ile Gly Thr Cys Phe 20 25 30 Gly Pro Arg Val Pro Cys Cys Arg Arg 35
40 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 67
<211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:
Bos taurus <400> SEQUENCE: 67 Gln Gly Val Arg Asn His Val Thr
Cys Arg Ile Asn Arg Gly Phe Cys 1 5 10 15 Val Pro Ile Arg Cys Pro
Gly Arg Thr Arg Gln Ile Gly Thr Cys Phe 20 25 30 Gly Pro Arg Ile
Lys Cys Cys Arg Ser Trp 35 40 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 68 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 68 Gln
Gly Val Arg Ser Tyr Leu Ser Cys Trp Gly Asn Arg Gly Ile Cys 1 5 10
15 Leu Leu Asn Arg Cys Pro Gly Arg Met Arg Gln Ile Gly Thr Cys Leu
20 25 30 Ala Pro Arg Val Lys Cys Cys Arg 35 40 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 69 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Bos taurus <400>
SEQUENCE: 69 Ser Gly Ile Ser Gly Pro Leu Ser Cys Gly Arg Asn Gly
Gly Val Cys 1 5 10 15 Ile Pro Ile Arg Cys Pro Val Pro Met Arg Gln
Ile Gly Thr Cys Phe 20 25 30 Gly Arg Pro Val Lys Cys Cys Arg Ser
Trp 35 40 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 70 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Bos taurus <400> SEQUENCE: 70 Asp Phe Ala Ser Cys
His Thr Asn Gly Gly Ile Cys Leu Pro Asn Arg 1 5 10 15 Cys Pro Gly
His Met Ile Gln Ile Gly Ile Cys Phe Arg Pro Arg Val 20 25 30 Lys
Cys Cys Arg Ser Trp 35 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 71 <211> LENGTH: 74 <212> TYPE:
PRT <213> ORGANISM: Zophobas atratus <400> SEQUENCE: 71
Ser Leu Gln Gly Gly Ala Pro Asn Phe Pro Gln Pro Ser Gln Gln Asn 1 5
10 15 Gly Gly Trp Gln Val Ser Pro Asp Leu Gly Arg Asp Asp Lys Gly
Asn 20 25 30 Thr Arg Gly Gln Ile Glu Ile Gln Asn Lys Gly Lys Asp
His Asp Phe 35 40 45 Asn Ala Gly Trp Gly Lys Val Ile Arg Gly Pro
Asn Lys Ala Lys Pro 50 55 60 Thr Trp His Val Gly Gly Thr Tyr Arg
Arg 65 70 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 72 <211> LENGTH: 67 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 72 Met Arg Ile His Tyr
Leu Leu Phe Ala Leu Leu Phe Leu Phe Leu Val 1 5 10 15 Pro Val Pro
Gly His Gly Gly Ile Ile Asn Thr Leu Gln Lys Tyr Tyr 20 25 30 Cys
Arg Val Arg Gly Gly Arg Cys Ala Val Leu Ser Cys Leu Pro Lys 35 40
45 Glu Glu Gln Ile Gly Lys Cys Ser Thr Arg Gly Arg Lys Cys Cys Arg
50 55 60 Arg Lys Lys 65 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 73 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Aedes aegypti <400> SEQUENCE: 73
Ala Thr Cys Asp Leu Leu Ser Gly Phe Gly Val Gly Asp Ser Ala Cys 1 5
10 15 Ala Ala His Cys Ile Ala Arg Gly Asn Arg Gly Gly Tyr Cys Asn
Ser 20 25 30
Lys Lys Val Cys Val Cys Arg Asn 35 40 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 74 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Mytilus edulis
<220> FEATURE: <221> NAME/KEY: SITE <222>
LOCATION: (28) <223> OTHER INFORMATION: Xaa at this position
can be any amino acid. <400> SEQUENCE: 74 Gly Phe Gly Cys Pro
Asn Asp Tyr Pro Cys His Arg His Cys Lys Ser 1 5 10 15 Ile Pro Gly
Arg Tyr Gly Gly Tyr Cys Gly Gly Xaa His Arg Leu Arg 20 25 30 Cys
Thr Cys 35 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 75 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Sarcophaga peregrina <400> SEQUENCE: 75 Ala Thr Cys
Asp Leu Leu Ser Gly Ile Gly Val Gln His Ser Ala Cys 1 5 10 15 Ala
Leu His Cys Val Phe Arg Gly Asn Arg Gly Gly Tyr Cys Thr Gly 20 25
30 Lys Gly Ile Cys Val Cys Arg Asn 35 40 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 76 <211> LENGTH: 95
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 76 Met Arg Thr Leu Ala Leu Leu Ala Ala Ile
Leu Leu Val Ala Leu Gln 1 5 10 15 Ala Gln Ala Glu His Val Ser Val
Ser Ile Asp Glu Val Val Asp Gln 20 25 30 Gln Pro Pro Gln Ala Glu
Asp Gln Asp Val Ala Ile Tyr Val Lys Glu 35 40 45 His Glu Ser Ser
Ala Leu Glu Ala Leu Gly Val Lys Ala Gly Val Val 50 55 60 Cys Ala
Cys Arg Arg Ala Leu Cys Leu Pro Arg Glu Arg Arg Ala Gly 65 70 75 80
Phe Cys Arg Ile Arg Gly Arg Ile His Pro Leu Cys Cys Arg Arg 85 90
95 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 77
<211> LENGTH: 92 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 77 Met Lys Pro Leu Val Leu Leu
Ser Ala Leu Val Leu Leu Ser Phe Gln 1 5 10 15 Val Gln Ala Asp Pro
Ile Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu 20 25 30 Glu Gln Ser
Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp 35 40 45 Arg
Glu Gly Ala Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys 50 55
60 Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg Glu Arg Met Asn Gly Thr
65 70 75 80 Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys 85 90
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 78
<211> LENGTH: 93 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 78 Met Lys Thr Phe Val Leu Leu
Ser Ala Leu Val Leu Leu Ala Phe Gln 1 5 10 15 Val Gln Ala Asp Pro
Ile His Lys Thr Asp Glu Glu Thr Asn Thr Glu 20 25 30 Glu Gln Pro
Gly Glu Glu Asp Gln Ala Val Ser Ile Ser Phe Gly Gly 35 40 45 Gln
Glu Gly Ser Ala Leu His Glu Glu Leu Ser Lys Lys Leu Ile Cys 50 55
60 Tyr Cys Arg Ile Arg Gly Cys Lys Arg Arg Glu Arg Val Phe Gly Thr
65 70 75 80 Cys Arg Asn Leu Phe Leu Thr Phe Val Phe Cys Cys Ser 85
90 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 79
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 79 Leu Arg Asp Leu Val Cys Tyr
Cys Arg Ala Arg Gly Cys Lys Gly Arg 1 5 10 15 Glu Arg Met Asn Gly
Thr Cys Arg Lys Gly His Leu Leu Tyr Met Leu 20 25 30 Cys Cys Arg 35
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 80
<211> LENGTH: 43 <212> TYPE: PRT <213> ORGANISM:
Pyrrhocoris apterus <400> SEQUENCE: 80 Ala Thr Cys Asp Ile
Leu Ser Phe Gln Ser Gln Trp Val Thr Pro Asn 1 5 10 15 His Ala Gly
Cys Ala Leu His Cys Val Ile Lys Gly Tyr Lys Gly Gly 20 25 30 Gln
Cys Lys Ile Thr Val Cys His Cys Arg Arg 35 40 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 81 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus
<400> SEQUENCE: 81 Val Thr Cys Tyr Cys Arg Ser Thr Arg Cys
Gly Phe Arg Glu Arg Leu 1 5 10 15 Ser Gly Ala Cys Gly Tyr Arg Gly
Arg Ile Tyr Arg Leu Cys Cys Arg 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 82 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus
<400> SEQUENCE: 82 Val Thr Cys Ser Cys Arg Thr Ser Ser Cys
Arg Phe Gly Glu Arg Leu 1 5 10 15 Ser Gly Ala Cys Arg Leu Asn Gly
Arg Ile Tyr Arg Leu Cys Cys 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 83 <211> LENGTH: 34
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 83 Gly Ile Cys Ala Cys Arg Arg Arg Phe Cys
Leu Asn Phe Glu Gln Phe 1 5 10 15 Ser Gly Tyr Cys Arg Val Asn Gly
Ala Arg Tyr Val Arg Cys Cys Ser 20 25 30 Arg Arg <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 84 <211>
LENGTH: 64 <212> TYPE: PRT <213> ORGANISM: Pan
troglodytes <400> SEQUENCE: 84 Met Arg Val Leu Tyr Leu Leu
Phe Ser Phe Leu Phe Ile Phe Leu Met 1 5 10 15 Pro Leu Pro Gly Val
Phe Gly Gly Ile Ser Asp Pro Val Thr Cys Leu 20 25 30 Lys Ser Gly
Ala Ile Cys His Pro Val Phe Cys Pro Arg Arg Tyr Lys 35 40 45 Gln
Ile Gly Thr Cys Gly Leu Pro Gly Thr Lys Cys Cys Lys Lys Pro 50 55
60 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 85
<211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 85 Met Arg Val Leu Tyr Leu Leu
Phe Ser Phe Leu Phe Ile Phe Leu Met 1 5 10 15 Pro Leu Pro Gly Val
Phe Gly Gly Ile Gly Asp Pro Val Thr Cys Leu 20 25 30 Lys Ser Gly
Ala Ile Cys His Pro Val Phe Cys Pro Arg Arg Tyr Lys 35 40 45 Gln
Ile Gly Thr Cys Gly Leu Pro Gly Thr Lys Cys Cys Lys Lys Pro 50 55
60 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 86
<211> LENGTH: 68 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 86 Met Arg Thr Ser Tyr Leu Leu
Leu Phe Thr Leu Cys Leu Leu Leu Ser 1 5 10 15 Glu Met Ala Ser Gly
Gly Asn Phe Leu Thr Gly Leu Gly His Arg Ser 20 25 30 Asp His Tyr
Asn Cys Val Ser Ser Gly Gly Gln Cys Leu Tyr Ser Ala 35 40 45 Cys
Pro Ile Phe Thr Lys Ile Gln Gly Thr Cys Tyr Arg Gly Lys Ala 50 55
60 Lys Cys Cys Lys 65 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 87 <211> LENGTH: 64 <212> TYPE:
PRT <213> ORGANISM: Capra hircus <400> SEQUENCE: 87 Met
Arg Leu His His Leu Leu Leu Val Leu Phe Phe Leu Val Leu Ser 1 5 10
15 Ala Gly Ser Gly Phe Thr Gln Gly Ile Arg Ser Arg Arg Ser Cys His
20 25 30 Arg Asn Lys Gly Val Cys Ala Leu Thr Arg Cys Pro Arg Asn
Met Arg 35 40 45 Gln Ile Gly Thr Cys Phe Gly Pro Pro Val Lys Cys
Cys Arg Lys Lys 50 55 60 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 88 <211> LENGTH: 64 <212> TYPE:
PRT <213> ORGANISM: Capra hircus <400> SEQUENCE: 88 Met
Arg Leu His His Leu Leu Leu Ala Leu Phe Phe Leu Val Leu Ser 1 5 10
15 Ala Gly Ser Gly Phe Thr Gln Gly Ile Ile Asn His Arg Ser Cys Tyr
20 25 30 Arg Asn Lys Gly Val Cys Ala Pro Ala Arg Cys Pro Arg Asn
Met Arg 35 40 45 Gln Ile Gly Thr Cys His Gly Pro Pro Val Lys Cys
Cys Arg Lys Lys 50 55 60 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 89 <211> LENGTH: 96 <212> TYPE:
PRT <213> ORGANISM: Macaca mulatta <400> SEQUENCE: 89
Met Arg Thr Leu Val Ile Leu Ala Ala Ile Leu Leu Val Ala Leu Gln 1 5
10 15 Ala Gln Ala Glu Pro Leu Gln Ala Arg Thr Asp Glu Ala Thr Ala
Ala 20 25 30 Gln Glu Gln Ile Pro Thr Asp Asn Pro Glu Val Val Val
Ser Leu Ala 35 40 45 Trp Asp Glu Ser Leu Ala Pro Lys Asp Ser Val
Pro Gly Leu Arg Lys 50 55 60 Asn Met Ala Cys Tyr Cys Arg Ile Pro
Ala Cys Leu Ala Gly Glu Arg 65 70 75 80 Arg Tyr Gly Thr Cys Phe Tyr
Arg Arg Arg Val Trp Ala Phe Cys Cys 85 90 95 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 90 <211> LENGTH: 96
<212> TYPE: PRT <213> ORGANISM: Macaca mulatta
<400> SEQUENCE: 90 Met Arg Thr Leu Val Ile Leu Ala Ala Ile
Leu Leu Val Ala Leu Gln 1 5 10 15 Ala Gln Ala Glu Pro Leu Gln Ala
Arg Thr Asp Glu Ala Thr Ala Ala 20 25 30 Gln Glu Gln Ile Pro Thr
Asp Asn Pro Glu Val Val Val Ser Leu Ala 35 40 45 Trp Asp Glu Ser
Leu Ala Pro Lys Asp Ser Val Pro Gly Leu Arg Lys 50 55 60 Asn Met
Ala Cys Tyr Cys Arg Ile Pro Ala Cys Leu Ala Gly Glu Arg 65 70 75 80
Arg Tyr Gly Thr Cys Phe Tyr Leu Gly Arg Val Trp Ala Phe Cys Cys 85
90 95 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
91 <211> LENGTH: 33 <212> TYPE: PRT
<213> ORGANISM: Mesocricetus auratus <400> SEQUENCE: 91
Val Thr Cys Phe Cys Arg Arg Arg Gly Cys Ala Ser Arg Glu Arg His 1 5
10 15 Ile Gly Tyr Cys Arg Phe Gly Asn Thr Ile Tyr Arg Leu Cys Cys
Arg 20 25 30 Arg <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 92 <211> LENGTH: 31 <212> TYPE: PRT
<213> ORGANISM: Mesocricetus auratus <400> SEQUENCE: 92
Cys Phe Cys Lys Arg Pro Val Cys Asp Ser Gly Glu Thr Gln Ile Gly 1 5
10 15 Tyr Cys Arg Leu Gly Asn Thr Phe Tyr Arg Leu Cys Cys Arg Gln
20 25 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 93 <211> LENGTH: 39 <212> TYPE: PRT <213>
ORGANISM: Gallus gallus <400> SEQUENCE: 93 Gly Arg Lys Ser
Asp Cys Phe Arg Lys Asn Gly Phe Cys Ala Phe Leu 1 5 10 15 Lys Cys
Pro Tyr Leu Thr Leu Ile Ser Gly Lys Cys Ser Arg Phe His 20 25 30
Leu Cys Cys Lys Arg Ile Trp 35 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 94 <211> LENGTH: 43
<212> TYPE: PRT <213> ORGANISM: Allomyrina dichotoma
<400> SEQUENCE: 94 Val Thr Cys Asp Leu Leu Ser Phe Glu Ala
Lys Gly Phe Ala Ala Asn 1 5 10 15 His Ser Leu Cys Ala Ala His Cys
Leu Ala Ile Gly Arg Arg Gly Gly 20 25 30 Ser Cys Glu Arg Gly Val
Cys Ile Cys Arg Arg 35 40 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 95 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Cavia porcellus <400> SEQUENCE: 95
Arg Arg Cys Ile Cys Thr Thr Arg Thr Cys Arg Phe Pro Tyr Arg Arg 1 5
10 15 Leu Gly Thr Cys Ile Phe Gln Asn Arg Val Tyr Thr Phe Cys Cys
20 25 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 96 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence Synthetic
<400> SEQUENCE: 96 Xaa Cys Asn Cys Arg Asn Cys Asn Glu Arg
Asn Cys Asn Gly Asn Cys 1 5 10 15 Cys Xaa
* * * * *